Compositions and methods for the therapy and diagnosis of ovarian cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly ovarian cancer, are disclosed. Illustrative compositions comprise one or more ovarian tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly ovarian cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/713,550, filed Nov. 14, 2000, which is a CIP ofSer. No. 09/656,668, filed Sep. 7, 2000, which is a CIP of U.S.application Ser. No. 09/640,173, filed Aug. 15, 2000, which is a is aCIP of U.S. application Ser. No. 09/561,778, filed May 1, 2000, which isa CIP of U.S. application Ser. No. 09/394,374, filed Sep. 10, 1999, allpending and incorporated by reference in their entirety herein.

TECHNICAL FIELD

[0002] The present invention relates generally to ovarian cancertherapy. The invention is more specifically related to polypeptidescomprising at least a portion of an ovarian carcinoma protein, and topolynucleotides encoding such polypeptides, as well as antibodies andimmune system cells that specifically recognize such polypeptides. Suchpolypeptides, polynucleotides, antibodies and cells may be used invaccines and pharmaceutical compositions for treatment of ovariancancer.

BACKGROUND OF THE INVENTION

[0003] Ovarian cancer is a significant health problem for women in theUnited States and throughout the world. Although advances have been madein detection and therapy of this cancer, no vaccine or other universallysuccessful method for prevention or treatment is currently available.Management of the disease currently relies on a combination of earlydiagnosis and aggressive treatment, which may include one or more of avariety of treatments such as surgery, radiotherapy, chemotherapy andhormone therapy. The course of treatment for a particular cancer isoften selected based on a variety of prognostic parameters, including ananalysis of specific tumor markers. However, the use of establishedmarkers often leads to a result that is difficult to interpret, and highmortality continues to be observed in many cancer patients.

[0004] Immunotherapies have the potential to substantially improvecancer treatment and survival. Such therapies may involve the generationor enhancement of an immune response to an ovarian carcinoma antigen.However, to date, relatively few ovarian carcinoma antigens are knownand the generation of an immune response against such antigens has notbeen shown to be therapeutically beneficial.

[0005] Accordingly, there is a need in the art for improved methods foridentifying ovarian tumor antigens and for using such antigens in thetherapy of ovarian cancer. The present invention fulfills these needsand further provides other related advantages.

SUMMARY OF THE INVENTION

[0006] Briefly stated, this invention provides compositions and methodsfor the therapy of cancer, such as ovarian cancer.

[0007] In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

[0008] (a) sequences provided in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19,23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75,78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121,125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162,166-168, 171, 174-183, 185, 193-199, 203-206, and 208;

[0009] (b) complements of the sequences provided in SEQ ID NO:1, 2, 5,9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57,63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111,114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151,156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, and208;

[0010] (c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28,32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84,86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128,132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168,171, 174-183, 185, 193-199, 203-206, and 208;

[0011] (d) sequences that hybridize to a sequence provided in SEQ IDNO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52,53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100,103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140,143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, and 208 under moderately stringent conditions;

[0012] (e) sequences having at least 75% identity to a sequence providedin SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38,41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95,97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137,140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208, and 210-214;

[0013] (f) sequences having at least 90% identity to a sequence providedin SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38,41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95,97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137,140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208 and 210-214; and

[0014] (g) degenerate variants of a sequence provided in SEQ ID NO:1, 2,5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57,63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111,114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151,156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and210-214.

[0015] In one preferred embodiment, the polynucleotide compositions ofthe invention are expressed in at least about 20%, more preferably in atleast about 30%, and most preferably in at least about 50% of ovariantumors samples tested, at a level that is at least about 2-fold,preferably at least about 5-fold, and most preferably at least about10-fold higher than that for normal tissues.

[0016] In one aspect, the present invention provides polypeptidescomprising an immunogenic portion of an ovarian carcinoma protein, or avariant thereof that differs in one or more substitutions, deletions,additions and/or insertions such that the ability of the variant toreact with ovarian carcinoma protein-specific antisera is notsubstantially diminished. Within certain embodiments, the ovariancarcinoma protein comprises a sequence that is encoded by apolynucleotide sequence selected from the group consisting of SEQ IDNO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52,53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100,103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140,143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-205, 208 and 210-214, and complements of suchpolynucleotides.

[0017] The present invention further provides polynucleotides thatencode a polypeptide as described above or a portion thereof, expressionvectors comprising such polynucleotides and host cells transformed ortransfected with such expression vectors.

[0018] The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO:200-202, 207, 209 and 215.

[0019] In certain preferred embodiments, the polypeptides of the presentinvention are immunogenic, i.e., they are capable of eliciting an immuneresponse, particularly a humoral and/or cellular immune response, asfurther described herein.

[0020] The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide sequences, wherein thefragments, variants and/or derivatives preferably have a level ofimmunogenic activity of at least about 50%, preferably at least about70% and more preferably at least about 90% of the level of immunogenicactivity of a the ovarian carcinoma protein comprises an amino acidsequence encoded by a polynucleotide that comprises a sequence recitedin any one of SEQ ID NO:1-185, 187-199, 203-206, 208 and 210-214.

[0021] Within other aspects, the present invention providespharmaceutical compositions comprising a polypeptide and/orpolynucleotide as described above and a physiologically acceptablecarrier.

[0022] Within a related aspect of the present invention, thepharmaceutical compositions, e.g., vaccine compositions, are providedfor prophylactic or therapeutic applications. Such compositionsgenerally comprise an immunogenic polypeptide or polynucleotide of theinvention and an immunostimulant, such as an adjuvant.

[0023] The present invention further provides pharmaceuticalcompositions that comprise: (a) an antibody or antigen-binding fragmentthereof that specifically binds to a polypeptide of the presentinvention, or a fragment thereof, and (b) a physiologically acceptablecarrier.

[0024] Within further aspects, the present invention providespharmaceutical compositions comprising: (a) an antigen presenting cellthat expresses a polypeptide as described above and (b) apharmaceutically acceptable carrier or excipient. Illustrative antigenpresenting cells include dendritic cells, macrophages, monocytes,fibroblasts and B cells.

[0025] Within related aspects, pharmaceutical compositions are providedthat comprise: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) an immunostimulant.

[0026] The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

[0027] Within further aspects, the present invention provides methodsfor stimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with ovariancancer, in which case the methods provide treatment for the disease, orpatient considered at risk for such a disease may be treatedprophylactically.

[0028] Within further aspects, the present invention provides methodsfor inhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with ovarian cancer, in which casethe methods provide treatment for the disease, or patient considered atrisk for such a disease may be treated prophylactically.

[0029] The present invention further provides, within other aspects,methods for removing tumor cells from a biological sample, comprisingcontacting a biological sample with T cells that specifically react witha polypeptide of the present invention, wherein the step of contactingis performed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

[0030] Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

[0031] Methods are further provided, within other aspects, forstimulating and/or expanding T cells specific for a polypeptide of thepresent invention, comprising contacting T cells with one or more of:(i) an ovarian carcinoma polypeptide as described above; (ii) apolynucleotide encoding such a polypeptide; and/or (iii) an antigenpresenting cell that expresses such a polypeptide; under conditions andfor a time sufficient to permit the stimulation and/or expansion of Tcells. Isolated T cell populations comprising T cells prepared asdescribed above are also provided.

[0032] Within further aspects, the present invention provides methodsfor inhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

[0033] The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺and/or CD8⁺T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofpolypeptide disclosed herein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, and thereby inhibiting the development of acancer in the patient. Proliferated cells may, but need not, be clonedprior to administration to the patient.

[0034] Within further aspects, the present invention provides methodsfor determining the presence or absence of a cancer, preferably anovarian cancer, in a patient comprising: (a) contacting a biologicalsample obtained from a patient with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; and (c) comparing theamount of polypeptide with a predetermined cut-off value, and therefromdetermining the presence or absence of a cancer in the patient. Withinpreferred embodiments, the binding agent is an antibody, more preferablya monoclonal antibody.

[0035] The present invention also provides, within other aspects,methods for monitoring the progression of a cancer in a patient. Suchmethods comprise the steps of: (a) contacting a biological sampleobtained from a patient at a first point in time with a binding agentthat binds to a polypeptide as recited above; (b) detecting in thesample an amount of polypeptide that binds to the binding agent; (c)repeating steps (a) and (b) using a biological sample obtained from thepatient at a subsequent point in time; and (d) comparing the amount ofpolypeptide detected in step (c) with the amount detected in step (b)and therefrom monitoring the progression of the cancer in the patient.

[0036] The present invention further provides, within other aspects,methods for determining the presence or absence of a cancer in apatient, comprising the steps of: (a) contacting a biological sampleobtained from a patient with an oligonucleotide that hybridizes to apolynucleotide that encodes a polypeptide of the present invention; (b)detecting in the sample a level of a polynucleotide, preferably mRNA,that hybridizes to the oligonucleotide; and (c) comparing the level ofpolynucleotide that hybridizes to the oligonucleotide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within certain embodiments, theamount of mRNA is detected via polymerase chain reaction using, forexample, at least one oligonucleotide primer that hybridizes to apolynucleotide encoding a polypeptide as recited above, or a complementof such a polynucleotide. Within other embodiments, the amount of mRNAis detected using a hybridization technique, employing anoligonucleotide probe that hybridizes to a polynucleotide that encodes apolypeptide as recited above, or a complement of such a polynucleotide.

[0037] In related aspects, methods are provided for monitoring theprogression of a cancer in a patient, comprising the steps of: (a)contacting a biological sample obtained from a patient with anoligonucleotide that hybridizes to a polynucleotide that encodes apolypeptide of the present invention; (b) detecting in the sample anamount of a polynucleotide that hybridizes to the oligonucleotide; (c)repeating steps (a) and (b) using a biological sample obtained from thepatient at a subsequent point in time; and (d) comparing the amount ofpolynucleotide detected in step (c) with the amount detected in step (b)and therefrom monitoring the progression of the cancer in the patient.

[0038] Within further aspects, the present invention providesantibodies, such as monoclonal antibodies, that bind to a polypeptide asdescribed above, as well as diagnostic kits comprising such antibodies.Diagnostic kits comprising one or more oligonucleotide probes or primersas described above are also provided.

[0039] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description. Allreferences disclosed herein are hereby incorporated by reference intheir entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

[0040] SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38,41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95,97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137,140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,and 193-199 are described in Tables III-VII below.

[0041] SEQ ID NO:200 is the amino acid sequence of a polypeptide encodedby the polynucleotide recited in SEQ ID NO: 182;

[0042] SEQ ID NO:201 is the amino acid sequence of a polypeptide encodedby the polynucleotide recited in SEQ ID NO: 182;

[0043] SEQ ID NO:202 is the amino acid sequence of a polypeptide encodedby the polynucleotide recited in SEQ ID NO:182.

[0044] SEQ ID NO:203 is the determined extended cDNA sequence for SEQ IDNO:197.

[0045] SEQ ID NO:204 is the determined extended cDNA sequence for SEQ IDNO:198.

[0046] SEQ ID NO:205 is the determined extended cDNA sequence for SEQ IDNO:199.

[0047] SEQ ID NO:206 is the determined cDNA sequence for the codingregion of O568S fused to an N-terminal His tag.

[0048] SEQ ID NO:207 is the amino acid sequence of the polypeptideencoded by the polynucleotide recited in SEQ ID NO:206.

[0049] SEQ ID NO:208 is the determined cDNA sequence for the codingregion of GPR39 as downloaded from the High Throughput GenomicsDatabase.

[0050] SEQ ID NO:209 is the amino acid sequence encoded by the cDNAsequence recited in SEQ ID NO:208.

[0051] SEQ ID NO:210 is the nucleotide sequence od O1034C an ovaryspecific EST clone discovered using electronic subtraction.

[0052] SEQ ID NO:211 is the full length nucleotide sequence of O591 S.

[0053] SEQ ID NO:212 is the sequence BF345141 which shows sequencehomology with O1034C/O591 S allowing for the extension of O591 S.

[0054] SEQ ID NO:213 is the sequence BE336607 which shows sequencehomology with O1034C/O591S allowing for the extension of O591S.

[0055] SEQ ID NO:214 is the consensus nucleotide sequence ofO1034C/O591S containing 1897 base pairs.

[0056] SEQ ID NO:215 is the predicted translation of the open readingframe identified within SEQ ID NO:214 (nucleotides 260-682).

DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention is directed generally to compositions andtheir use in the therapy and diagnosis of cancer, particularly ovariancancer. As described further below, illustrative compositions of thepresent invention include, but are not restricted to, polypeptides,particularly immunogenic polypeptides, polynucleotides encoding suchpolypeptides, antibodies and other binding agents, antigen presentingcells (APCs) and immune system cells (e.g., T cells).

[0058] The practice of the present invention will employ, unlessindicated specifically to the contrary, conventional methods ofvirology, immunology, microbiology, molecular biology and recombinantDNA techniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

[0059] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0060] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

Polypeptide Compositions

[0061] As used herein, the term “polypeptide” “is used in itsconventional meaning, i.e., as a sequence of amino acids. Thepolypeptides are not limited to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

[0062] Particularly illustrative polypeptides of the present inventioncomprise those encoded by a polynucleotide sequence set forth in any oneof SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38,41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95,97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137,140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208, and 210-214 or a sequence that hybridizes undermoderately stringent conditions, or, alternatively, under highlystringent conditions, to a polynucleotide sequence identified above.Certain other illustrative polypeptides of the invention comprise aminoacid sequences as set forth in any one of SEQ ID NO:200-202, 207, 209and 215.

[0063] The polypeptides of the present invention are sometimes hereinreferred to as ovarian tumor proteins or ovarian tumor polypeptides, asan indication that their identification has been based at least in partupon their increased levels of expression in ovarian tumor samples.Thus, a “ovarian tumor polypeptide” or “ovarian tumor protein,” refersgenerally to a polypeptide sequence of the present invention, or apolynucleotide sequence encoding such a polypeptide, that is expressedin a substantial proportion of ovarian tumor samples, for examplepreferably greater than about 20%, more preferably greater than about30%, and most preferably greater than about 50% or more of ovarian tumorsamples tested, at a level that is at least two fold, and preferably atleast five fold, greater than the level of expression in normal tissues,as determined using a representative assay provided herein. An ovariantumor polypeptide sequence of the invention, based upon its increasedlevel of expression in tumor cells, has particular utility both as adiagnostic marker as well as a therapeutic target, as further describedbelow.

[0064] In certain preferred embodiments, the polypeptides of theinvention are immunogenic, i.e., they react detectably within animmunoassay (such as an ELISA or T-cell stimulation assay) with antiseraand/or T-cells from a patient with ovarian cancer. Screening forimmunogenic activity can be performed using techniques well known to theskilled artisan. For example, such screens can be performed usingmethods such as those described in Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988. In oneillustrative example, a polypeptide may be immobilized on a solidsupport and contacted with patient sera to allow binding of antibodieswithin the sera to the immobilized polypeptide. Unbound sera may then beremoved and bound antibodies detected using, for example, ¹²⁵I-labeledProtein A.

[0065] As would be recognized by the skilled artisan, immunogenicportions of the polypeptides disclosed herein are also encompassed bythe present invention. An “immunogenic portion,” as used herein, is afragment of an immunogenic polypeptide of the invention that itself isimmunologically reactive (i.e., specifically binds) with the B-cellsand/or T-cell surface antigen receptors that recognize the polypeptide.Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

[0066] In one preferred embodiment, an immunogenic portion of apolypeptide of the present invention is a portion that reacts withantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full-length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Preferably, the level of immunogenic activityof the immunogenic portion is at least about 50%, preferably at leastabout 70% and most preferably greater than about 90% of theimmunogenicity for the fill-length polypeptide. In some instances,preferred immunogenic portions will be identified that have a level ofimmunogenic activity greater than that of the corresponding full-lengthpolypeptide, e.g., having greater than about 100% or 150% or moreimmunogenic activity.

[0067] In certain other embodiments, illustrative immunogenic portionsmay include peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

[0068] In another embodiment, a polypeptide composition of the inventionmay also comprise one or more polypeptides that are immunologicallyreactive with T cells and/or antibodies generated against a polypeptideof the invention, particularly a polypeptide having an amino acidsequence disclosed herein, or to an immunogenic fragment or variantthereof.

[0069] In another embodiment of the invention, polypeptides are providedthat comprise one or more polypeptides that are capable of eliciting Tcells and/or antibodies that are immunologically reactive with one ormore polypeptides described herein, or one or more polypeptides encodedby contiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

[0070] The present invention, in another aspect, provides polypeptidefragments comprising at least about 5, 10, 15, 20, 25, 50, or 100contiguous amino acids, or more, including all intermediate lengths, ofa polypeptide compositions set forth herein, such as those set forth inSEQ ID NO:200-202, 207, 209, and 215 or those encoded by apolynucleotide sequence set forth in any one of SEQ ID NO:1, 2, 5, 9,10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63,65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114,117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156,158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208, and210-214.

[0071] In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

[0072] In one preferred embodiment, the polypeptide fragments andvariants provide by the present invention are immunologically reactivewith an antibody and/or T-cell that reacts with a full-lengthpolypeptide specifically set for the herein.

[0073] In another preferred embodiment, the polypeptide fragments andvariants provided by the present invention exhibit a level ofimmunogenic activity of at least about 50%, preferably at least about70%, and most preferably at least about 90% or more of that exhibited bya full-length polypeptide sequence specifically set forth herein.

[0074] A polypeptide “variant,” as the term is used herein, is apolypeptide that typically differs from a polypeptide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of theabove polypeptide sequences of the invention and evaluating theirimmunogenic activity as described herein and/or using any of a number oftechniques well known in the art.

[0075] For example, certain illustrative variants of the polypeptides ofthe invention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

[0076] In many instances, a variant will contain conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. As described above, modifications may be madein the structure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

[0077] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated thatvarious changes may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity. TABLE1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGGUGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAG AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0078] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (4.5).

[0079] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e. still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 (specifically incorporated herein by reference in itsentirety), states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

[0080] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0081] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0082] In addition, any polynucleotide may be further modified toincrease stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

[0083] Amino acid substitutions may further be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

[0084] As noted above, polypeptides may comprise a signal (or leader)sequence at the N-terminal end of the protein, which co-translationallyor post-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

[0085] When comparing polypeptide sequences, two sequences are said tobe “identical” if the sequence of amino acids in the two sequences isthe same when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

[0086] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0087] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0088] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

[0089] In one preferred approach, the “percentage of sequence identity”is determined by comparing two optimally aligned sequences over a windowof comparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

[0090] Within other illustrative embodiments, a polypeptide may be afusion polypeptide that comprises multiple polypeptides as describedherein, or that comprises at least one polypeptide as described hereinand an unrelated sequence, such as a known tumor protein. A fusionpartner may, for example, assist in providing T helper epitopes (animmunological fusion partner), preferably T helper epitopes recognizedby humans, or may assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant protein. Certainpreferred fusion partners are both immunological and expressionenhancing fusion partners. Other fusion partners may be selected so asto increase the solubility of the polypeptide or to enable thepolypeptide to be targeted to desired intracellular compartments. Stillfurther fusion partners include affinity tags, which facilitatepurification of the polypeptide.

[0091] Fusion polypeptides may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusionpolypeptide is expressed as a recombinant polypeptide, allowing theproduction of increased levels, relative to a non-fused polypeptide, inan expression system. Briefly, DNA sequences encoding the polypeptidecomponents may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion polypeptide that retains the biologicalactivity of both component polypeptides.

[0092] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusionpolypeptide using standard techniques well known in the art. Suitablepeptide linker sequences may be chosen based on the following factors:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes. Preferred peptide linker sequencescontain Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala may also be used in the linker sequence. Amino acidsequences which may be usefully employed as linkers include thosedisclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc.Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 andU.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 toabout 50 amino acids in length. Linker sequences are not required whenthe first and second polypeptides have non-essential N-terminal aminoacid regions that can be used to separate the functional domains andprevent steric interference.

[0093] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

[0094] The fusion polypeptide can comprise a polypeptide as describedherein together with an unrelated immunogenic protein, such as animmunogenic protein capable of eliciting a recall response. Examples ofsuch proteins include tetanus, tuberculosis and hepatitis proteins (see,for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

[0095] In one preferred embodiment, the immunological fusion partner isderived from a Mycobacterium sp., such as a Mycobacteriumtuberculosis-derived Ra12 fragment. Ra12 compositions and methods fortheir use in enhancing the expression and/or immunogenicity ofheterologous polynucleotide/polypeptide sequences is described in U.S.patent application Ser. No. 60/158,585, the disclosure of which isincorporated herein by reference in its entirety. Briefly, Ra12 refersto a polynucleotide region that is a subsequence of a Mycobacteriumtuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KDmolecular weight encoded by a gene in virulent and avirulent strains ofM. tuberculosis. The nucleotide sequence and amino acid sequence ofMTB32A have been described (for example, U.S. patent application Ser.No. 60/158,585; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). C-terminal fragments ofthe MTB32A coding sequence express at high levels and remain as asoluble polypeptides throughout the purification process. Moreover, Ra12 may enhance the immunogenicity of heterologous immunogenicpolypeptides with which it is fused. One preferred Ra12 fusionpolypeptide comprises a 14 KD C-terminal fragment corresponding to aminoacid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotidesgenerally comprise at least about 15 consecutive nucleotides, at leastabout 30 nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

[0096] Within other preferred embodiments, an immunological fusionpartner is derived from protein D, a surface protein of thegram-negative bacterium Haemophilus influenza B (WO 91/18926).Preferably, a protein D derivative comprises approximately the firstthird of the protein (e.g., the first N-terminal 100-110 amino acids),and a protein D derivative may be lipidated. Within certain preferredembodiments, the first 109 residues of a Lipoprotein D fusion partner isincluded on the N-terminus to provide the polypeptide with additionalexogenous T-cell epitopes and to increase the expression level in E.coli (thus functioning as an expression enhancer). The lipid tailensures optimal presentation of the antigen to antigen presenting cells.Other fusion partners include the non-structural protein from influenzaevirus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids areused, although different fragments that include T-helper epitopes may beused.

[0097] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terminalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0098] Yet another illustrative embodiment involves fusion polypeptides,and the polynucleotides encoding them, wherein the fusion partnercomprises a targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation ofCD4⁺T-cells specific for the polypeptide.

[0099] Polypeptides of the invention are prepared using any of a varietyof well known synthetic and/or recombinant techniques, the latter ofwhich are further described below. Polypeptides, portions and othervariants generally less than about 150 amino acids can be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. In one illustrative example, such polypeptides aresynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

[0100] In general, polypeptide compositions (including fusionpolypeptides) of the invention are isolated. An “isolated” polypeptideis one that is removed from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

[0101] The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

[0102] As will be understood by those skilled in the art, thepolynucleotide compositions of this invention can include genomicsequences, extra-genomic and plasmid-encoded sequences and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, peptides and the like. Such segments may benaturally isolated, or modified synthetically by the hand of man.

[0103] As will be also recognized by the skilled artisan,polynucleotides of the invention may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules may include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

[0104] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes a polypeptide/protein of the inventionor a portion thereof) or may comprise a sequence that encodes a variantor derivative, preferably and immunogenic variant or derivative, of sucha sequence.

[0105] Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NO:1, 2, 5, 9,10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63,65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114,117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156,158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and210-214, complements of a polynucleotide sequence set forth as describedabove, and degenerate variants of a polynucleotide sequence set forth asdescribed above. In certain preferred embodiments, the polynucleotidesequences set forth herein encode immunogenic polypeptides, as describedabove.

[0106] In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28,32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84,86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128,132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168,171, 174-183, 185, 193-199, 203-206, 208 and 210-214, for example thosecomprising at least 70% sequence identity, preferably at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identitycompared to a polynucleotide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

[0107] Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

[0108] In additional embodiments, the present invention providespolynucleotide fragments comprising various lengths of contiguousstretches of sequence identical to or complementary to one or more ofthe sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 10, 15, 20, 30,40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

[0109] In another embodiment of the invention, polynucleotidecompositions are provided that are capable of hybridizing under moderateto high stringency conditions to a polynucleotide sequence providedherein, or a fragment thereof, or a complementary sequence thereof.Hybridization techniques are well known in the art of molecular biology.For purposes of illustration, suitable moderately stringent conditionsfor testing the hybridization of a polynucleotide of this invention withother polynucleotides include prewashing in a solution of 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC,overnight; followed by washing twice at 65° C. for 20 minutes with eachof 2×, 0.5× and 0.2×SSC containing 0.1% SDS. One skilled in the art willunderstand that the stringency of hybridization can be readilymanipulated, such as by altering the salt content of the hybridizationsolution and/or the temperature at which the hybridization is performed.For example, in another embodiment, suitable highly stringenthybridization conditions include those described above, with theexception that the temperature of hybridization is increased, e.g., to60-65° C. or 65-70° C.

[0110] In certain preferred embodiments, the polynucleotides describedabove, e.g., polynucleotide variants, fragments and hybridizingsequences, encode polypeptides that are immunologically cross-reactivewith a polypeptide sequence specifically set forth herein. In otherpreferred embodiments, such polynucleotides encode polypeptides thathave a level of immunogenic activity of at least about 50%, preferablyat least about 70%, and more preferably at least about 90% of that for apolypeptide sequence specifically set forth herein.

[0111] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrativepolynucleotide segments with total lengths of about 10,000, about 5000,about 3000, about 2,000, about 1,000, about 500, about 200, about 100,about 50 base pairs in length, and the like, (including all intermediatelengths) are contemplated to be useful in many implementations of thisinvention.

[0112] When comparing polynucleotide sequences, two sequences are saidto be “identical” if the sequence of nucleotides in the two sequences isthe same when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

[0113] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0114] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0115] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

[0116] Preferably, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (ie., the windowsize) and multiplying the results by 100 to yield the percentage ofsequence identity.

[0117] It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

[0118] Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

[0119] Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

[0120] In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

[0121] As will be appreciated by those of skill in the art,site-specific mutagenesis techniques have often employed a phage vectorthat exists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage are readily commercially-available and their useis generally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

[0122] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0123] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis provides ameans of producing potentially useful species and is not meant to belimiting as there are other ways in which sequence variants of peptidesand the DNA sequences encoding them may be obtained. For example,recombinant vectors encoding the desired peptide sequence may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. Specific details regarding these methods and protocols arefound in the teachings of Maloy et al., 1994; Segal, 1976; Prokop andBajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporatedherein by reference, for that purpose.

[0124] As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

[0125] In another approach for the production of polypeptide variants ofthe present invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

[0126] In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

[0127] The ability of such nucleic acid probes to specifically hybridizeto a sequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

[0128] Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

[0129] The use of a hybridization probe of about 15-25 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

[0130] Hybridization probes may be selected from any portion of any ofthe sequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

[0131] Small polynucleotide segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.No. 4,683,202 (incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

[0132] The nucleotide sequences of the invention may be used for theirability to selectively form duplex molecules with complementarystretches of the entire gene or gene fragments of interest. Depending onthe application envisioned, one will typically desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofprobe towards target sequence. For applications requiring highselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids, e.g., one will select relatively lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating relatedsequences.

[0133] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0134] According to another embodiment of the present invention,polynucleotide compositions comprising antisense oligonucleotides areprovided. Antisense oligonucleotides have been demonstrated to beeffective and targeted inhibitors of protein synthesis, and,consequently, provide a therapeutic approach by which a disease can betreated by inhibiting the synthesis of proteins that contribute to thedisease. The efficacy of antisense oligonucleotides for inhibitingprotein synthesis is well established. For example, the synthesis ofpolygalactauronase and the muscarine type 2 acetylcholine receptor areinhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDG1),ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF(Jaskulski et al., Science. 1988 June 10;240(4858):1544-6; Vasanthakumarand Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res MolBrain Res. 1998 June 15;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat.No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288).Antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

[0135] Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, Tm, binding energy, and relativestability. Antisense compositions may be selected based upon theirrelative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0136] The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 July15;25(14):2730-6). It has been demonstrated that several molecules ofthe MPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

[0137] According to another embodiment of the invention, thepolynucleotide compositions described herein are used in the design andpreparation of ribozyme molecules for inhibiting expression of the tumorpolypeptides and proteins of the present invention in tumor cells.Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA.1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 April24;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December;27(3 Pt 2):487-96; Micheland Westhof, J Mol Biol. 1990 December 5;216(3):585-610; Reinhold-Hurekand Shub, Nature. 1992 May 14;357(6374):173-6). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

[0138] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

[0139] The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al, Proc Natl Acad SciUSA. 1992 August 15;89(16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

[0140] The enzymatic nucleic acid molecule may be formed in ahammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA(in association with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 September 1 1;20(17):4559-65. Examples of hairpin motifsare described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 June 13;28(12):4929-33; Hampel etal., Nucleic Acids Res. 1990 January 25;18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 December 1;31(47): 11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. 1990 May18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991October 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 March23;32(11):2795-9); and an example of the Group I intron is described in(U.S. Pat. 4,987,071). All that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target geneRNA regions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

[0141] Ribozymes may be designed as described in Int. Pat. Appl. Publ.No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, eachspecifically incorporated herein by reference) and synthesized to betested in vitro and in vivo, as described. Such ribozymes can also beoptimized for delivery. While specific examples are provided, those inthe art will recognize that equivalent RNA targets in other species canbe utilized when necessary.

[0142] Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g, Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No. WO93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ.No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ. No.WO 94/13688, which describe various chemical modifications that can bemade to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

[0143] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describesthe general methods for delivery of enzymatic RNA molecules. Ribozymesmay be administered to cells by a variety of methods known to thosefamiliar to the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

[0144] Another means of accumulating high concentrations of aribozyme(s) within cells is to incorporate the ribozyme-encodingsequences into a DNA expression vector. Transcription of the ribozymesequences are driven from a promoter for eukaryotic RNA polymerase I(pol I), RNA polymerase II (pol TI), or RNA polymerase III (pol III).Transcripts from pol II or pol III promoters will be expressed at highlevels in all cells; the levels of a given pol II promoter in a givencell type will depend on the nature of the gene regulatory sequences(enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerasepromoters may also be used, providing that the prokaryotic RNApolymerase enzyme is expressed in the appropriate cells Ribozymesexpressed from such promoters have been shown to function in mammaliancells. Such transcription units can be incorporated into a variety ofvectors for introduction into mammalian cells, including but notrestricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated vectors), or viral RNA vectors (such asretroviral, semliki forest virus, sindbis virus vectors).

[0145] In another embodiment of the invention, peptide nucleic acids(PNAs) compositions are provided. PNA is a DNA mimic in which thenucleobases are attached to a pseudopeptide backbone (Good and Nielsen,Antisense Nucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (Trends Biotechnol 1997June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

[0146] PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science 1991 December6;254(5037):1497-500; Hanvey et al., Science. 1992 November27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996January;4(1):5-23). This chemistry has three important consequences:firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAsare neutral molecules; secondly, PNAs are achiral, which avoids the needto develop a stereoselective synthesis; and thirdly, PNA synthesis usesstandard Boc or Fmoc protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used.

[0147] PNA monomers or ready-made oligomers are commercially availablefrom PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by eitherBoc or Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April;3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

[0148] As with peptide synthesis, the success of a particular PNAsynthesis will depend on the properties of the chosen sequence. Forexample, while in theory PNAs can incorporate any combination ofnucleotide bases, the presence of adjacent purines can lead to deletionsof one or more residues in the product. In expectation of thisdifficulty, it is suggested that, in producing PNAs with adjacentpurines, one should repeat the coupling of residues likely to be addedinefficiently. This should be followed by the purification of PNAs byreverse-phase high-pressure liquid chromatography, providing yields andpurity of product similar to those observed during the synthesis ofpeptides.

[0149] Modifications of PNAs for a given application may be accomplishedby coupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April;3(4):437-45; Petersen et al., J PeptSci. 1995 May-June;1(3):175-83; Orum et al, Biotechniques. 1995September;19(3):472-80; Footer et al., Biochemistry. 1996 August20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 August11;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. 1995 June6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. 1995 March14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Augus15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 November11;94(23):12320-5; Seeger et al., Biotechniques. 1997September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNAchimeric molecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

[0150] Methods of characterizing the antisense binding properties ofPNAs are discussed in Rose (Anal Chem. 1993 December 15;65(24):3545-9)and Jensen et al. (Biochemistry. 1997 April 22;36(16):5072-7). Rose usescapillary gel electrophoresis to determine binding of PNAs to theircomplementary oligonucleotide, measuring the relative binding kineticsand stoichiometry. Similar types of measurements were made by Jensen etal. using BIAcore™ technology.

[0151] Other applications of PNAs that have been described and will beapparent to the skilled artisan include use in DNA strand invasion,antisense inhibition, mutational analysis, enhancers of transcription,nucleic acid purification, isolation of transcriptionally active genes,blocking of transcription factor binding, genome cleavage, biosensors,in situ hybridization, and the like.

Polynucleotide Identification, Characterization and Expression

[0152] Polynucleotides compositions of the present invention may beidentified, prepared and/or manipulated using any of a variety of wellestablished techniques (see generally, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989, and other like references). For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using the microarraytechnology of Affymetrix, Inc. (Santa Clara, Calif.) according to themanufacturers instructions (and essentially as described by Schena etal., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al.,Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Alternatively,polynucleotides may be amplified from cDNA prepared from cellsexpressing the proteins described herein, such as tumor cells.

[0153] Many template dependent processes are available to amplify atarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

[0154] Any of a number of other template dependent processes, many ofwhich are variations of the PCR™ amplification technique, are readilyknown and available in the art. Illustratively, some such methodsinclude the ligase chain reaction (referred to as LCR), described, forexample, in Eur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No.4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair ChainReaction (RCR). Still other amplification methods are described in GreatBritain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

[0155] An amplified portion of a polynucleotide of the present inventionmay be used to isolate a full length gene from a suitable library (e.g.,a tumor cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

[0156] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

[0157] Alternatively, amplification techniques, such as those describedabove, can be useful for obtaining a full length coding sequence from apartial cDNA sequence. One such amplification technique is inverse PCR(see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which usesrestriction enzymes to generate a fragment in the known region of thegene. The fragment is then circularized by intramolecular ligation andused as a template for PCR with divergent primers derived from the knownregion. Within an alternative approach, sequences adjacent to a partialsequence may be retrieved by amplification with a primer to a linkersequence and a primer specific to a known region. The amplifiedsequences are typically subjected to a second round of amplificationwith the same linker primer and a second primer specific to the knownregion. A variation on this procedure, which employs two primers thatinitiate extension in opposite directions from the known sequence, isdescribed in WO 96/38591. Another such technique is known as “rapidamplification of cDNA ends” or RACE. This technique involves the use ofan internal primer and an external primer, which hybridizes to a polyAregion or vector sequence, to identify sequences that are 5′ and 3′ of aknown sequence. Additional techniques include capture PCR (Lagerstrom etal., PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

[0158] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

[0159] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode polypeptides of the invention, orfusion proteins or functional equivalents thereof, may be used inrecombinant DNA molecules to direct expression of a polypeptide inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide.

[0160] As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

[0161] Moreover, the polynucleotide sequences of the present inventioncan be engineered using methods generally known in the art in order toalter polypeptide encoding sequences for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the gene product. For example, DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

[0162] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

[0163] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of a polypeptide, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 43 1A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0164] A newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.) or other comparable techniques available in theart. The composition of the synthetic peptides may be confirmed by aminoacid analysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

[0165] In order to express a desired polypeptide, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into appropriate expression vector, ie., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods which are well known to thoseskilled in the art may be used to construct expression vectorscontaining sequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.

[0166] A variety of expression vector/host systems may be utilized tocontain and express polynucleotide sequences. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0167] The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

[0168] In bacterial systems, any of a number of expression vectors maybe selected depending upon the use intended for the expressedpolypeptide. For example, when large quantities are needed, for examplefor the induction of antibodies, vectors which direct high levelexpression of fusion proteins that are readily purified may be used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the polypeptide of interest may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of .beta.-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0169] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0170] In cases where plant expression vectors are used, the expressionof sequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0171] An insect system may also be used to express a polypeptide ofinterest. For example, in one such system, Autographa califomica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

[0172] In mammalian host cells, a number of viral-based expressionsystems are generally available. For example, in cases where anadenovirus is used as an expression vector, sequences encoding apolypeptide of interest may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0173] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding a polypeptide of interest.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

[0174] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0175] For long-term, high-yield production of recombinant proteins,stable expression is generally preferred. For example, cell lines whichstably express a polynucleotide of interest may be transformed usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

[0176] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990)Cell 22:817-23) genes which can be employed in tk.sup.- oraprt.sup.-cells, respectively. Also, antimetabolite, antibiotic orherbicide resistance can be used as the basis for selection; forexample, dhfr which confers resistance to methotrexate (Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confersresistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin,F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Murry, supra). Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. 85:8047-51). The use of visible markers has gained popularitywith such markers as anthocyanins, beta-glucuronidase and its substrateGUS, and luciferase and its substrate luciferin, being widely used notonly to identify transformants, but also to quantify the amount oftransient or stable protein expression attributable to a specific vectorsystem (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0177] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0178] Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

[0179] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide may bepreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

[0180] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0181] Host cells transformed with a polynucleotide sequence of interestmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides ofthe invention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

[0182] In addition to recombinant production methods, polypeptides ofthe invention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents

[0183] According to another aspect, the present invention furtherprovides binding agents, such as antibodies and antigen-bindingfragments thereof, that exhibit immunological binding to a tumorpolypeptide disclosed herein, or to a portion, variant or derivativethereof. An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunogically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably with unrelated polypeptides under similarconditions.

[0184] Immunological binding, as used in this context, generally refersto the non-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

[0185] An “antigen-binding site,” or “binding portion” of an antibodyrefers to the part of the immunoglobulin molecule that participates inantigen binding. The antigen binding site is formed by amino acidresidues of the N-terminal variable (“V”) regions of the heavy (“H”) andlight (“L”) chains. Three highly divergent stretches within the Vregions of the heavy and light chains are referred to as “hypervariableregions” which are interposed between more conserved flanking stretchesknown as “framework regions,” or “FRs”. Thus the term “FR” refers toamino acid sequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

[0186] Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as ovarian cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

[0187] Any agent that satisfies the above requirements may be a bindingagent. For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

[0188] Monoclonal antibodies specific for an antigenic polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0189] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

[0190] A number of therapeutically useful molecules are known in the artwhich comprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂ ” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0191] A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

[0192] Each of the above-described molecules includes a heavy chain anda light chain CDR set, respectively interposed between a heavy chain anda light chain FR set which provide support to the CDRS and define thespatial relationship of the CDRs relative to each other. As used herein,the term “CDR set” refers to the three hypervariable regions of a heavyor light chain V region. Proceeding from the N-terminus of a heavy orlight chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

[0193] As used herein, the term “FR set” refers to the four flankingamino acid sequences which frame the CDRs of a CDR set of a heavy orlight chain V region. Some FR residues may contact bound antigen;however, FRs are primarily responsible for folding the V region into theantigen-binding site, particularly the FR residues directly adjacent tothe CDRS. Within FRs, certain amino residues and certain structuralfeatures are very highly conserved. In this regard, all V regionsequences contain an internal disulfide loop of around 90 amino acidresidues. When the V regions fold into a binding-site, the CDRs aredisplayed as projecting loop motifs which form an antigen-bindingsurface. It is generally recognized that there are conserved structuralregions of FRs which influence the folded shape of the CDR loops intocertain “canonical” structures—regardless of the precise CDR amino acidsequence. Further, certain FR residues are known to participate innon-covalent interdomain contacts which stabilize the interaction of theantibody heavy and light chains.

[0194] A number of “humanized” antibody molecules comprising anantigen-binding site derived from a non-human immunoglobulin have beendescribed, including chimeric antibodies having rodent V regions andtheir associated CDRs fused to human constant domains (Winter et al.(1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci.USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brownet al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into ahuman supporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

[0195] As used herein, the terms “veneered FRs” and “recombinantlyveneered FRs” refer to the selective replacement of FR residues from,e.g., a rodent heavy or light chain V region, with human FR residues inorder to provide a xenogeneic molecule comprising an antigen-bindingsite which retains substantially all of the native FR polypeptidefolding structure. Veneering techniques are based on the understandingthat the ligand binding characteristics of an antigen-binding site aredetermined primarily by the structure and relative disposition of theheavy and light chain CDR sets within the antigen-binding surface.Davies et al. (1990) Ann. Rev. Biochem. 59:439-473. Thus, antigenbinding specificity can be preserved in a humanized antibody onlywherein the CDR structures, their interaction with each other, and theirinteraction with the rest of the V region domains are carefullymaintained. By using veneering techniques, exterior (e.g.,solvent-accessible) FR residues which are readily encountered by theimmune system are selectively replaced with human residues to provide ahybrid molecule that comprises either a weakly immunogenic, orsubstantially non-immunogenic veneered surface.

[0196] The process of veneering makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al., inSequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. ofHealth and Human Services, U.S. Government Printing Office, 1987),updates to the Kabat database, and other accessible U.S. and foreigndatabases (both nucleic acid and protein). Solvent accessibilities of Vregion amino acids can be deduced from the known three-dimensionalstructure for human and murine antibody fragments. There are two generalsteps in veneering a murine antigen-binding site. Initially, the FRs ofthe variable domains of an antibody molecule of interest are comparedwith corresponding FR sequences of human variable domains obtained fromthe above-identified sources. The most homologous human V regions arethen compared residue by residue to corresponding murine amino acids.The residues in the murine FR which differ from the human counterpartare replaced by the residues present in the human moiety usingrecombinant techniques well known in the art. Residue switching is onlycarried out with moieties which are at least partially exposed (solventaccessible), and care is exercised in the replacement of amino acidresidues which may have a significant effect on the tertiary structureof V region domains, such as proline, glycine and charged amino acids.

[0197] In this manner, the resultant “veneered” murine antigen-bindingsites are thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

[0198] In another embodiment of the invention, monoclonal antibodies ofthe present invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

[0199] A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

[0200] Alternatively, it may be desirable to couple a therapeutic agentand an antibody via a linker group. A linker group can function as aspacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on an agent or anantibody, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of agents, or functionalgroups on agents, which otherwise would not be possible.

[0201] It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

[0202] Where a therapeutic agent is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

[0203] It may be desirable to couple more than one agent to an antibody.In one embodiment, multiple molecules of an agent are coupled to oneantibody molecule. In another embodiment, more than one type of agentmay be coupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

[0204] A carrier may bear the agents in a variety of ways, includingcovalent bonding either directly or via a linker group. Suitablecarriers include proteins such as albumins (e.g., U.S. Pat. No.4,507,234, to Kato et al.), peptides and polysaccharides such asaminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carriermay also bear an agent by noncovalent bonding or by encapsulation, suchas within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and4,873,088). Carriers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al.discloses representative chelating compounds and their synthesis.

T Cell Compositions

[0205] The present invention, in another aspect, provides T cellsspecific for a tumor polypeptide disclosed herein, or for a variant orderivative thereof. Such cells may generally be prepared in vitro or exvivo, using standard procedures. For example, T cells may be isolatedfrom bone marrow, peripheral blood, or a fraction of bone marrow orperipheral blood of a patient, using a commercially available cellseparation system, such as the Isolex™ System, available from NexellTherapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856;U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).Alternatively, T cells may be derived from related or unrelated humans,non-human mammals, cell lines or cultures.

[0206] T cells may be stimulated with a polypeptide, polynucleotideencoding a polypeptide and/or an antigen presenting cell (APC) thatexpresses such a polypeptide. Such stimulation is performed underconditions and for a time sufficient to permit the generation of T cellsthat are specific for the polypeptide of interest. Preferably, a tumorpolypeptide or polynucleotide of the invention is present within adelivery vehicle, such as a microsphere, to facilitate the generation ofspecific T cells.

[0207] T cells are considered to be specific for a polypeptide of thepresent invention if the T cells specifically proliferate, secretecytokines or kill target cells coated with the polypeptide or expressinga gene encoding the polypeptide. T cell specificity may be evaluatedusing any of a variety of standard techniques. For example, within achromium release assay or proliferation assay, a stimulation index ofmore than two fold increase in lysis and/or proliferation, compared tonegative controls, indicates T cell specificity. Such assays may beperformed, for example, as described in Chen et al., Cancer Res.54:1065-1070, 1994. Alternatively, detection of the proliferation of Tcells may be accomplished by a variety of known techniques. For example,T cell proliferation can be detected by measuring an increased rate ofDNA synthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Contact with a tumor polypeptide (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically resultin at least a two fold increase in proliferation of the T cells. Contactas described above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

[0208] For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferatein response to a tumor polypeptide, polynucleotide or APC can beexpanded in number either in vitro or in vivo. Proliferation of such Tcells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

Pharmaceutical Compositions

[0209] In additional embodiments, the present invention concernsformulation of one or more of the polynucleotide, polypeptide, T-celland/or antibody compositions disclosed herein inpharmaceutically-acceptable carriers for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy.

[0210] It will be understood that, if desired, a composition asdisclosed herein may be administered in combination with other agents aswell, such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

[0211] Therefore, in another aspect of the present invention,pharmaceutical compositions are provided comprising one or more of thepolynucleotide, polypeptide, antibody, and/or T-cell compositionsdescribed herein in combination with a physiologically acceptablecarrier. In certain preferred embodiments, the pharmaceuticalcompositions of the invention comprise immunogenic polynucleotide and/orpolypeptide compositions of the invention for use in prophylactic andtheraputic vaccine applications. Vaccine preparation is generallydescribed in, for example, M. F. Powell and M. J. Newman, eds., “VaccineDesign (the subunit and adjuvant approach),” Plenum Press (NY, 1995).Generally, such compositions will comprise one or more polynucleotideand/or polypeptide compositions of the present invention in combinationwith one or more immunostimulants.

[0212] It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

[0213] In another embodiment, illustrative immunogenic compositions,e.g., vaccine compositions, of the present invention comprise DNAencoding one or more of the polypeptides as described above, such thatthe polypeptide is generated in situ. As noted above, the polynucleotidemay be administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

[0214] Therefore, in certain embodiments, polynucleotides encodingimmunogenic polypeptides described herein are introduced into suitablemammalian host cells for expression using any of a number of knownviral-based systems. In one illustrative embodiment, retrovirusesprovide a convenient and effective platform for gene delivery systems. Aselected nucleotide sequence encoding a polypeptide of the presentinvention can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of illustrativeretroviral systems have been described (e.g., U.S. Pat. No. 5,219,740;Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990)Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; andBoris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0215] In addition, a number of illustrative adenovirus-based systemshave also been described. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

[0216] Various adeno-associated virus (AAV) vector systems have alsobeen developed for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0217] Additional viral vectors useful for delivering thepolynucleotides encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0218] A vaccinia-based infection/transfection system can beconveniently used to provide for inducible, transient expression orcoexpression of one or more polypeptides described herein in host cellsof an organism. In this particular system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide orpolynucleotides of interest, driven by a T7 promoter. The polymeraseexpressed in the cytoplasm from the vaccinia virus recombinanttranscribes the transfected DNA into RNA which is then translated intopolypeptide by the host translational machinery. The method provides forhigh level, transient, cytoplasmic production of large quantities of RNAand its translation products. See, e.g., Elroy-Stein and Moss, Proc.Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl.Acad. Sci. USA (1986) 83:8122-8126.

[0219] Alternatively, avipoxviruses, such as the fowlpox and canarypoxviruses, can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0220] Any of a number of alphavirus vectors can also be used fordelivery of polynucleotide compositions of the present invention, suchas those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686;6,008,035 and 6,015,694. Certain vectors based on Venezuelan EquineEncephalitis (VEE) can also be used, illustrative examples of which canbe found in U.S. Pat. Nos. 5,505,947 and 5,643,576.

[0221] Moreover, molecular conjugate vectors, such as the adenoviruschimeric vectors described in Michael et al. J. Biol. Chem. (1993)268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992)89:6099-6103, can also be used for gene delivery under the invention.

[0222] Additional illustrative information on these and other knownviral-based delivery systems can be found, for example, in Fisher-Hochet al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al.,Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973;U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219,1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502,1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al.,Cir. Res. 73:1202-1207, 1993.

[0223] In certain embodiments, a polynucleotide may be integrated intothe genome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

[0224] In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

[0225] In still another embodiment, a composition of the presentinvention can be delivered via a particle bombardment approach, many ofwhich have been described. In one illustrative example, gas-drivenparticle acceleration can be achieved with devices such as thosemanufactured by Powderject Pharmaceuticals PLC (Oxford, UK) andPowderject Vaccines Inc. (Madison, Wis.), some examples of which aredescribed in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807;and EP Pat. No. 0500 799. This approach offers a needle-free deliveryapproach wherein a dry powder formulation of microscopic particles, suchas polynucleotide or polypeptide particles, are accelerated to highspeed within a helium gas jet generated by a hand held device,propelling the particles into a target tissue of interest.

[0226] In a related embodiment, other devices and methods that may beuseful for gas-driven needle-less, injection of compositions of thepresent invention include those provided by Bioject, Inc. (Portland,Oreg.), some examples of which are described in U.S. Pat. Nos.4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and5,993,412.

[0227] According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell and/orAPC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

[0228] Within certain embodiments of the invention, the adjuvantcomposition is preferably one that induces an immune responsepredominantly of the Th1 type. High levels of Th1-type cytokines (e.g.,IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cellmediated immune responses to an administered antigen. In contrast, highlevels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend tofavor the induction of humoral immune responses. Following applicationof a vaccine as provided herein, a patient will support an immuneresponse that includes Th1- and Th2-type responses. Within a preferredembodiment, in which a response is predominantly Th1-type, the level ofTh1-type cytokines will increase to a greater extent than the level ofTh2-type cytokines. The levels of these cytokines may be readilyassessed using standard assays. For a review of the families ofcytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

[0229] Certain preferred adjuvants for eliciting a predominantlyTh1-type response include, for example, a combination of monophosphoryllipid A, preferably 3-de-O-acylated monophosphoryl lipid A, togetherwith an aluminum salt. MPL® adjuvants are available from CorixaCorporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (inwhich the CpG dinucleotide is unmethylated) also induce a predominantlyTh1 response. Such oligonucleotides are well known and are described,for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200and 5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins .Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

[0230] Alternatively the saponin formulations may be combined withvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

[0231] In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

[0232] Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

[0233] Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

[0234] Other preferred adjuvants include adjuvant molecules of thegeneral formula

HO(CH₂CH₂0)_(n)—A—R,  (I):

[0235] wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orPhenyl C₁₋₅₀ alkyl.

[0236] One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein Is is between 1 and 50, preferably 4-24, most preferably 9; theR component is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

[0237] The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

[0238] According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0239] Certain preferred embodiments of the present invention usedendritic cells or progenitors thereof as antigen-presenting cells.Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

[0240] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, tumor-infiltrating cells, peritumoraltissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cordblood or any other suitable tissue or fluid. For example, dendriticcells may be differentiated ex vivo by adding a combination of cytokinessuch as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytesharvested from peripheral blood. Alternatively, CD34 positive cellsharvested from peripheral blood, umbilical cord blood or bone marrow maybe differentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

[0241] Dendritic cells are conveniently categorized as “immature” and“mature” cells, which allows a simple way to discriminate between twowell characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD1 1) and costimulatory molecules (e.g, CD40, CD80,CD86 and 4-1BB).

[0242] APCs may generally be transfected with a polynucleotide of theinvention (or portion or other variant thereof) such that the encodedpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

[0243] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will typically vary depending on the modeof administration. Compositions of the present invention may beformulated for any appropriate manner of administration, including forexample, topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

[0244] Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

[0245] In another illustrative embodiment, biodegradable microspheres(e.g., polylactate polyglycolate) are employed as carriers for thecompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and5,942,252. Modified hepatitis B core protein carrier systems. such asdescribed in WO/99 40934, and references cited therein, will also beuseful for many applications. Another illustrative carrier/deliverysystem employs a carrier comprising particulate-protein complexes, suchas those described in U.S. Pat. No. 5,928,647, which are capable ofinducing a class I-restricted cytotoxic T lymphocyte responses in ahost.

[0246] The pharmaceutical compositions of the invention will oftenfurther comprise one or more buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, bacteriostats, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes thatrender the formulation isotonic, hypotonic or weakly hypertonic with theblood of a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

[0247] The pharmaceutical compositions described herein may be presentedin unit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

[0248] The development of suitable dosing and treatment regimens forusing the particular compositions described herein in a variety oftreatment regimens, including e.g., oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art, some of which are briefly discussed below for generalpurposes of illustration.

[0249] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0250] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (see, forexample, Mathiowitz et al., Nature 1997 March 27;386(6623):410-4; Hwanget al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0251] Typically, these formulations will contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0252] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation.

[0253] Alternatively, the active ingredient may be incorporated into anoral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

[0254] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515and 5,399,363. In certain embodiments, solutions of the active compoundsas free base or pharmacologically acceptable salts may be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations generally will contain apreservative to prevent the growth of microorganisms.

[0255] Illustrative pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0256] In one embodiment, for parenteral administration in an aqueoussolution, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. Moreover, for humanadministration, preparations will of course preferably meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

[0257] In another embodiment of the invention, the compositionsdisclosed herein may be formulated in a neutral or salt form.Illustrative pharmaceutically-acceptable salts include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective.

[0258] The carriers can further comprise any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

[0259] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 March 2;52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

[0260] In certain embodiments, liposomes, nanocapsules, microparticles,lipid particles, vesicles, and the like, are used for the introductionof the compositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

[0261] The formation and use of liposome and liposome-like preparationsas potential drug carriers is generally known to those of skill in theart (see for example, Lasic, Trends Biotechnol 1998 July;16(7):307-21;Takakura, Nippon Rinsho 1998 March;56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August;35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995;12(2-3):233-61; U.S. Pat. Nos. 5,567,434; 5,552,157;5,565,213; 5,738,868 and 5,795,587, each specifically incorporatedherein by reference in its entirety).

[0262] Liposomes have been used successfully with a number of cell typesthat are normally difficult to transfect by other procedures, includingT cell suspensions, primary hepatocyte cultures and PC 12 cells(Renneisen et al., J Biol Chem. 1990 September 25;265(27):16337-42;Muller et al., DNA Cell Biol. 1990 April;9(3):221-9). In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, various drugs, radiotherapeutic agents, enzymes,viruses, transcription factors, allosteric effectors and the like, intoa variety of cultured cell lines and animals. Furthermore, he use ofliposomes does not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

[0263] In certain embodiments, liposomes are formed from phospholipidsthat are dispersed in an aqueous medium and spontaneously formmultilamellar concentric bilayer vesicles (also termed multilamellarvesicles (MLVs).

[0264] Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December;24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst.1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 January2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

[0265] In further aspects of the present invention, the pharmaceuticalcompositions described herein may be used for the treatment of cancer,particularly for the immunotherapy of ovarian cancer. Within suchmethods, the pharmaceutical compositions described herein areadministered to a patient, typically a warm-blooded animal, preferably ahuman. A patient may or may not be afflicted with cancer. Accordingly,the above pharmaceutical compositions may be used to prevent thedevelopment of a cancer or to treat a patient afflicted with a cancer.Pharmaceutical compositions and vaccines may be administered eitherprior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. As discussed above, administration of thepharmaceutical compositions may be by any suitable method, includingadministration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

[0266] Within certain embodiments, immunotherapy may be activeimmunotherapy, in which treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against tumors with theadministration of immune response-modifying agents (such as polypeptidesand polynucleotides as provided herein).

[0267] Within other embodiments, immunotherapy may be passiveimmunotherapy, in which treatment involves the delivery of agents withestablished tumor-immune reactivity (such as effector cells orantibodies) that can directly or indirectly mediate antitumor effectsand does not necessarily depend on an intact host immune system.Examples of effector cells include T cells as discussed above, Tlymphocytes (such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helpertumor-infiltrating lymphocytes), killer cells (such as Natural Killercells and lymphokine-activated killer cells), B cells andantigen-presenting cells (such as dendritic cells and macrophages)expressing a polypeptide provided herein. T cell receptors and antibodyreceptors specific for the polypeptides recited herein may be cloned,expressed and transferred into other vectors or effector cells foradoptive immunotherapy. The polypeptides provided herein may also beused to generate antibodies or anti-idiotypic antibodies (as describedabove and in U.S. Pat. No. 4,918,164) for passive immunotherapy.

[0268] Effector cells may generally be obtained in sufficient quantitiesfor adoptive immunotherapy by growth in vitro, as described herein.Culture conditions for expanding single antigen-specific effector cellsto several billion in number with retention of antigen recognition invivo are well known in the art. Such in vitro culture conditionstypically use intermittent stimulation with antigen, often in thepresence of cytokines (such as IL-2) and non-dividing feeder cells. Asnoted above, immunoreactive polypeptides as provided herein may be usedto rapidly expand antigen-specific T cell cultures in order to generatea sufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

[0269] Alternatively, a vector expressing a polypeptide recited hereinmay be introduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

[0270] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and may be readily established using standardtechniques. In general, the pharmaceutical compositions and vaccines maybe administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

[0271] In general, an appropriate dosage and treatment regimen providesthe active compound(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit. Such a response can be monitored byestablishing an improved clinical outcome (e.g., more frequentremissions, complete or partial, or longer disease-free survival) intreated patients as compared to non-treated patients. Increases inpreexisting immune responses to a tumor protein generally correlate withan improved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which may be performed using samples obtained from a patient before andafter treatment.

Cancer Detection and Diagnostic Compositions, Methods and Kits

[0272] In general, a cancer may be detected in a patient based on thepresence of one or more ovarian tumor proteins and/or polynucleotidesencoding such proteins in a biological sample (for example, blood, sera,sputum urine and/or tumor biopsies) obtained from the patient. In otherwords, such proteins may be used as markers to indicate the presence orabsence of a cancer such as ovarian cancer. In addition, such proteinsmay be useful for the detection of other cancers. The binding agentsprovided herein generally permit detection of the level of antigen thatbinds to the agent in the biological sample. Polynucleotide primers andprobes may be used to detect the level of mRNA encoding an ovarian tumorprotein, which is also indicative of the presence or absence of acancer. In general, a ovarian tumor sequence should be present at alevel that is at least three fold higher in tumor tissue than in normaltissue

[0273] There are a variety of assay formats known to those of ordinaryskill in the art for using a binding agent to detect polypeptide markersin a sample. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In general, the presence orabsence of a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

[0274] In a preferred embodiment, the assay involves the use of bindingagent immobilized on a solid support to bind to and remove thepolypeptide from the remainder of the sample. The bound polypeptide maythen be detected using a detection reagent that contains a reportergroup and specifically binds to the binding agent/polypeptide complex.Such detection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length ovarian tumor proteins and polypeptide portions thereof towhich the binding agent binds, as described above.

[0275] The solid support may be any material known to those of ordinaryskill in the art to which the tumor protein may be attached. Forexample, the solid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

[0276] Covalent attachment of binding agent to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the binding agent. For example,the binding agent may be covalently attached to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe binding partner (see, e.g., Pierce Immunotechnology Catalog andHandbook, 1991, at A12-A13).

[0277] In certain embodiments, the assay is a two-antibody sandwichassay. This assay may be performed by first contacting an antibody thathas been immobilized on a solid support, commonly the well of amicrotiter plate, with the sample, such that polypeptides within thesample are allowed to bind to the immobilized antibody. Unbound sampleis then removed from the immobilized polypeptide-antibody complexes anda detection reagent (preferably a second antibody capable of binding toa different site on the polypeptide) containing a reporter group isadded. The amount of detection reagent that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

[0278] More specifically, once the antibody is immobilized on thesupport as described above, the remaining protein binding sites on thesupport are typically blocked. Any suitable blocking agent known tothose of ordinary skill in the art, such as bovine serum albumin orTween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibodyis then incubated with the sample, and polypeptide is allowed to bind tothe antibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with ovarian cancer. Preferably, the contacttime is sufficient to achieve a level of binding that is at least about95% of that achieved at equilibrium between bound and unboundpolypeptide. Those of ordinary skill in the art will recognize that thetime necessary to achieve equilibrium may be readily determined byassaying the level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

[0279] Unbound sample may then be removed by washing the solid supportwith an appropriate buffer, such as PBS containing 0.1% Tween 20™. Thesecond antibody, which contains a reporter group, may then be added tothe solid support. Preferred reporter groups include those groupsrecited above.

[0280] The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

[0281] To determine the presence or absence of a cancer, such as ovariancancer, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. In one preferred embodiment, thecut-off value for the detection of a cancer is the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is three standard deviations above the predetermined cut-off valueis considered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (1 00%-specificity) that correspond to eachpossible cut-off value for the diagnostic test result. The cut-off valueon the plot that is the closest to the upper left-hand comer (i.e., thevalue that encloses the largest area) is the most accurate cut-offvalue, and a sample generating a signal that is higher than the cut-offvalue determined by this method may be considered positive.Alternatively, the cut-off value may be shifted to the left along theplot, to minimize the false positive rate, or to the right, to minimizethe false negative rate. In general, a sample generating a signal thatis higher than the cut-off value determined by this method is consideredpositive for a cancer.

[0282] In a related embodiment, the assay is performed in a flow-throughor strip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

[0283] Of course, numerous other assay protocols exist that are suitablefor use with the tumor proteins or binding agents of the presentinvention. The above descriptions are intended to be exemplary only. Forexample, it will be apparent to those of ordinary skill in the art thatthe above protocols may be readily modified to use tumor polypeptides todetect antibodies that bind to such polypeptides in a biological sample.The detection of such tumor protein specific antibodies may correlatewith the presence of a cancer.

[0284] A cancer may also, or alternatively, be detected based on thepresence of T cells that specifically react with a tumor protein in abiological sample. Within certain methods, a biological samplecomprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubatedwith a tumor polypeptide, a polynucleotide encoding such a polypeptideand/or an APC that expresses at least an immunogenic portion of such apolypeptide, and the presence or absence of specific activation of the Tcells is detected. Suitable biological samples include, but are notlimited to, isolated T cells. For example, T cells may be isolated froma patient by routine techniques (such as by Ficoll/Hypaque densitygradient centrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withpolypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate anotheraliquot of a T cell sample in the absence of tumor polypeptide to serveas a control. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

[0285] As noted above, a cancer may also, or alternatively, be detectedbased on the level of mRNA encoding a tumor protein in a biologicalsample. For example, at least two oligonucleotide primers may beemployed in a polymerase chain reaction (PCR) based assay to amplify aportion of a tumor cDNA derived from a biological sample, wherein atleast one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a polynucleotide encoding the tumor protein. Theamplified cDNA is then separated and detected using techniques wellknown in the art, such as gel electrophoresis. Similarly,oligonucleotide probes that specifically hybridize to a polynucleotideencoding a tumor protein may be used in a hybridization assay to detectthe presence of polynucleotide encoding the tumor protein in abiological sample.

[0286] To permit hybridization under assay conditions, oligonucleotideprimers and probes should comprise an oligonucleotide sequence that hasat least about 60%, preferably at least about 75% and more preferably atleast about 90%, identity to a portion of a polynucleotide encoding atumor protein of the invention that is at least 10 nucleotides, andpreferably at least 20 nucleotides, in length. Preferably,oligonucleotide primers and/or probes hybridize to a polynucleotideencoding a polypeptide described herein under moderately stringentconditions, as defined above. Oligonucleotide primers and/or probeswhich may be usefully employed in the diagnostic methods describedherein preferably are at least 10-40 nucleotides in length. In apreferred embodiment, the oligonucleotide primers comprise at least 10contiguous nucleotides, more preferably at least 15 contiguousnucleotides, of a DNA molecule having a sequence as disclosed herein.Techniques for both PCR based assays and hybridization assays are wellknown in the art (see, for example, Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, StocktonPress, NY, 1989).

[0287] One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

[0288] In another embodiment, the compositions described herein may beused as markers for the progression of cancer. In this embodiment,assays as described above for the diagnosis of a cancer may be performedover time, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

[0289] Certain in vivo diagnostic assays may be performed directly on atumor. One such assay involves contacting tumor cells with a bindingagent. The bound binding agent may then be detected directly orindirectly via a reporter group. Such binding agents may also be used inhistological applications. Alternatively, polynucleotide probes may beused within such applications.

[0290] As noted above, to improve sensitivity, multiple tumor proteinmarkers may be assayed within a given sample. It will be apparent thatbinding agents specific for different proteins provided herein may becombined within a single assay. Further, multiple primers or probes maybe used concurrently. The selection of tumor protein markers may bebased on routine experiments to determine combinations that results inoptimal sensitivity. In addition, or alternatively, assays for tumorproteins provided herein may be combined with assays for other knowntumor antigens.

[0291] The present invention further provides kits for use within any ofthe above diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a tumor protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

[0292] Alternatively, a kit may be designed to detect the level of mRNAencoding a tumor protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a tumor protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding atumor protein.

[0293] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0294] Example 1

Identification of Representative Ovarian Carcinoma cDNA Sequences

[0295] Primary ovarian tumor and metastatic ovarian tumor cDNA librarieswere each constructed in kanamycin resistant pZErO™−2 vector(Invitrogen) from pools of three different ovarian tumor RNA samples.For the primary ovarian tumor library, the following RNA samples wereused: (1) a moderately differentiated papillary serous carcinoma of a 41year old, (2) a stage IIIC ovarian tumor and (3) a papillary serousadenocarcinoma for a 50 year old caucasian. For the metastatic ovariantumor library, the RNA samples used were omentum tissue from: (1) ametastatic poorly differentiated papillary adenocarcinoma with psammomabodies in a 73 year old, (2) a metastatic poorly differentiatedadenocarcinoma in a 74 year old and (3) a metastatic poorlydifferentiated papillary adenocarcinoma in a 68 year old.

[0296] The number of clones in each library was estimated by platingserial dilutions of unamplified libraries. Insert data were determinedfrom 32 primary ovarian tumor clones and 32 metastatic ovarian tumorclones. The library characterization results are shown in Table I. TABLEI Characterization of cDNA Libraries # Clones Clones with Insert SizeAve. Insert Library in Library Insert (%) Range (bp) Size (bp) PrimaryOvarian 1,258,000  97 175-8000 2356 Tumor Metastatic 1,788,000 100150-4300 1755 Ovarian Tumor

[0297] Four subtraction libraries were constructed in ampicillinresistant pcDNA3.1 vector (Invitrogen). Two of the libraries were fromprimary ovarian tumors and two were from metastatic ovarian tumors. Ineach case, the number of restriction enzyme cuts within inserts wasminimized to generate full length subtraction libraries. Thesubtractions were each done with slightly different protocols, asdescribed in more detail below. A. POTS 2 Library: Primary Ovarian TumorSubtraction Library Tracer 10 μg primary ovarian tumor library, digestedwith Not I Driver 35 μg normal pancreas in pcDNA3.1 (+) 20 μg normalPBMC in pcDNA3.1 (+) 10 μg normal skin in pcDNA3.1 (+) 35 μg normal bonemarrow in pZErO ™ -2 Digested with Bam HI/Xho I/Sca I

[0298] Two hybridizations were performed, and Not I-cut pcDNA3.1(+) wasthe cloning vector for the subtracted library. Sequence results forpreviously unidentified clones that were randomly picked from thesubtracted library are presented in Table II. TABLE II Ovarian CarcinomaSequences Sequence SEQ ID NO 21907  1 21909  2 21911  5 21920  9 21921 10 25099 143 25101 144 25103 145 25107 146 25111 148 25113 149 25115150 25116 151 25752 156 25757 158 25763 160 25769 161 25770 162

[0299] B. POTS 7 Library: Primary Ovarian Tumor Subtraction LibraryTracer   10 μg primary ovarian tumor library, digested with Not I Driver  35 μg normal pancreas in pcDNA3.1 (+)   20 μg normal PBMC in pcDNA3.1(+)   10 μg normal skin in pcDNA3.1 (+)   35 μg normal bone marrow inpZErO ™ -2 Digested with Bam HI/Xho I/Sca I ˜25 μg pZErO ™ -2, digestedwith BAM HI and Xho I

[0300] Two hybridizations were performed, and Not I-cut pcDNA3.1(+) wasthe cloning vector for the subtracted library. Sequence results forpreviously unidentified clones that were randomly picked from thesubtracted library are presented in Table III. TABLE III OvarianCarcinoma Sequences Sequence SEQ ID NO 24937 125 24940 128 24946 13224950 133 24951 134 24955 136 24956 137 25791 166 25796 167 25797 16825804 171

[0301] C. OS1D Library: Metastatic Ovarian Tumor Subtraction LibraryTracer   10 μg metastatic ovarian library in pZErO ™ -2, digested withNot I Driver 24.5 μg normal pancreas in pcDNA3.1   14 μg normal PBMC inpcDNA3.1   14 μg normal skin in pcDNA3.1 24.5 μg normal bone marrow inpZErO ™ -2   50 μg pZErO ™ -2, digested with BAM HI/Xho I/Sfu I

[0302] Three hybridizations were performed, and the last twohybridizations were done with an additional 15 μg of biotinylatedpZErO™−2 to remove contaminating pZErO™−2 vectors. The cloning vectorfor the subtracted library was pcDNA3.1/Not I cut. Sequence results forpreviously unidentified clones that were randomly picked from thesubtracted library are presented in Table IV. TABLE IV Ovarian CarcinomaSequences Sequence SEQ ID NO 23645.1 13 23660.1 16 23666.1 19 23679.1 2324635 57 24647 63 24651 65 24661 69 24663 70 24664 71 24670 72 24675 7524683 78

[0303] D. OS1F Library: Metastatic Ovarian Tumor Subtraction LibraryTracer   10 μg metastatic ovarian tumor library, digested with Not IDriver 12.8 μg normal pancreas in pcDNA3.1  7.3 μg normal PBMC inpcDNA3.1  7.3 μg normal skin in pcDNA3.1 12.8 μg normal bone marrow inpZErO ™ -2   25 μg pZErO ™ -2, digested with BAM HI/Xho I/Sfu I

[0304] One hybridization was performed. The cloning vector for thesubtracted library was pcDNA3.1/Not I cut. Sequence results forpreviously unidentified clones that were randomly picked from thesubtracted library are presented in Table V. TABLE V Ovarian CarcinomaSequences Sequence SEQ ID NO 24336 (79% with H. sapiens mitochondrialgenome 27 (consensus sequence)) 24337 28 24341 (91% Homo sapienschromosome 5, BAC clone 32 249h5 (LBNL H149) 24344 33 24348 35 24351 3824355 (91% Homo sapiens chromosome 17, clone 41 hCIT.91_J_4) 24356 4224357 (87% S. scrofa mRNA for UDP glucose 43 pyrophosphorylase) 24358 4424359 (78% Human mRNA for KIAA0111 gene, 45 complete cds) 24360 46 2436147 24362 (88% Homo sapiens Chromosome 16 BAC clone 48 CIT987SK-A-233A7)24363 (87% Homo sapiens eukaryotic translation 49 elongation factor 1alpha 1 (EBE1A1) 24364 (89% Human DNA sequence from PAC 27K14 on 50chromosome Xp11.3--Xp11.4) 24367 (89% Homo sapiens 12p13.3 BAC RCPI11-52 935C2) 24368 53 24690 81 24692 82 24694 84 24696 86 24699 89 24701 9024703 91 24704 (88% Homo sapiens chromosome 9, clone 92 hRPK.401 G_18)24705 93 24707 95 24709 97 24711 98 24713 99 24714 (91% Human DNAsequence from clone 125N5 100  on chromosome 6q26-27) 24717 (89% Homosapiens proliferation-associated gene 103  A (natural killer-enhancingfactor A) (PAGA) 24727 107  24732 111  24737 (84% Human ADP/ATPtranslocase mRNA) 114  24741 117  24745 120  24746 121 

[0305] The sequences in Table VI, which correspond to known sequences,were also identified in the above libraries. TABLE VI Ovarian CarcinomaSequences SEQ Identity ID NO Sequence Library H. sapiens DNA for musclenicotinic acetylcholine  3 21910 POTS2 receptor gene promotor, cloneICRFc105F02104 Homo sapiens complement component 3 (C3) gene,  4 21913POTS2 exons 1-30 Homo sapiens SWI/SNF related, matrix associated,  621914 POTS2 actin dependent regulator of chromatin, subfamily a, member4 (SMARCA4) Human ferritin Heavy subunit mRNA, complete cds.  7 21915POTS2 Homo sapiens CGI-151 protein mRNA, complete cds  8 21916 POTS2HUMGFIBPA Human growth hormone-dependent 12 23627.1 OS1D insulin-likegrowth factor-binding protein Homo sapiens ribosomal protein, large, P0(RPLP0) 14 23647.1 OS1D mRNA HUMTRPM2A Human TRPM-2 mRNA 15 23657.1 OS1DHUMMTA Homo sapiens mitochondrial DNA 17 23661.1 OS1D HSU78095 Homosapiens placental bikunin mRNA 18 23662.1 OS1D HUMT1227HC Human mRNA forTI-227H 20 23669.1 OS1D HUMMTCG Human mitochondrion 21 23673.1 OS1D Homosapiens FK506-binding protein 1A (12kD) 22 23677.1 OS1D (FKBP1A) mRNAHomo sapiens mRNA for zinc-finger DNA-binding 24 24333 OS1F protein,complete cds Homo sapiens mRNA; cDNA DKFZp564E1962 25 24334 OS1F (fromclone DKFZp564E1962) Homo sapiens tumor protein, translationally- 2624335 OS1F controlled 1 (TPT1) mRNA. Homo sapiens interleukin 1 receptoraccessory 29 24338 OS1F protein (IL1RAP) mRNA. Human mRNA for K1AA0026gene 30 24339 OS1F Homo sapiens K-Cl cotransporter KCC4 mRNA, 31 24340OS1F complete cds Homo sapiens nuclear chloride ion channel protein 3424345 OS1F (NCC27) mRNA Homo sapiens mRNA for DEPP (decidual protein 3624349 OS1F induced by progesterone) Homo sapiens atrophin-1 interactingprotein 4 (AIP4) 37 24350 OS1F mRNA Human collagenase type IV mRNA, 3′end. 39 24352 OS1F Human mRNA for T-cell cyclophilin 40 24354 OS1F Homosapiens tumor suppressing subtransferable 51 24366 OS1F candidate 1(TSSC1) Homo sapiens clone 24452 mRNA sequence 54 24374 OS1F Homosapiens eukaryotic translation elongation factor 55 24627 OS1D 1 alpha 1(EEF1A1) Genomic sequence from Human 9q34 56 24634 OS1D Humaninsulin-like growth factor-binding protein-3 58 24636 OS1D gene Humanribosomal protein L3 mRNA, 3′ end 59 24638 OS1D Homo sapiens annexin II(lipocortin II) (ANX2) 60 24640 OS1D mRNA Homo sapiens tubulin, alpha,ubiquitous (K-ALPHA- 61 24642 OS1D 1) Human non-histone chromosomalprotein HMG-14 62 24645 OS1D mRNA Homo sapiens ferritin, heavypolypeptide 1 (FTH1) 64 24648 OS1D Homo sapiens 12p13.3 PAC RPCI1-96H9(Roswell 66 24653 OS1D Park Cancer Institute Human PACLibrary) Homosapiens T cell-specific tyrosine kinase mRNA 67 24655 OS1D Homo sapienskeratin 18 (RRT18) mRNA 68 24657 OS1D Homo sapiens growth arrestspecific transcript 5 gene 73 24671 OS1D Homo sapiens ribosomal proteinS7 (RPS7) 74 24673 OS1D Homo sapiens mRNA; cDNA DKFZp564H182 76 24677OS1D Human TSC-22 protein mRNA 77 24679 OS1D Human mRNA for ribosomalprotein 79 24687 OS1D Genomic sequence from Human 13 80 24689 OS1F Homosapiens clone IMAGE 286356 83 24693 OS1F Homo sapiens v-fos FBJ murineosteosarcoma viral 85 24695 OS1F oncogene homolog(FOS) mRNA Homo sapienshypothetical 43.2 Kd protein mRNA 87 24697 OS1F Human heat shock protein27 (HSPB1) gene exons 1- 88 24698 OS1F 3 Homo sapienssenescence-associated epithelial 94 24706 OS1F membrane protein (SEMP1)Human ferritin H chain mRNA 96 24708 OS1F Homo sapiens mRNA for K1AA0287gene 101  24715 OS1F Homo sapiens CGI-08 protein mRNA 102  24716 OS1F H.sapiens CpG island DNA genomic Msel fragment, 104  24719 OS1F clone 84a5Human clone 23722 mRNA 105  24721 OS1F Homo sapiens zinc finger proteinslug (SLUG) gene 106  24722 OS1F Homo sapiens (clone L6) E-cadherin(CDH1) gene 108  24728 OS1F Homo sapiens ribosomal protein L13 (RPL13)109  24729 OS1F H. sapiens RNA for snRNP protein B 110  24730 OS1F Homosapiens mRNA; cDNA DKFZp434K114 112  24734 OS1F Homo sapiens cornichonprotein mRNA 113  24735 OS1F Homo sapiens keratin 8 (KRT8) mRNA 115 24739 OS1F Human DNA sequence from PAC 29K1 on 116  24740 OS1Fchromosome 6p21.3-22.2. Homo sapiens mRNA for KIAA0762 protein 118 24742 OS1F Human clones 23667 and 23775 zinc finger protein 119  24744OS1F mRNA Human H19 RNA gene, complete cds. 122  24933 POTS7 Humantriosephosphate isomerase mRNA, complete 123  24934 POTS7 cds. Humancyclooxygenase-1 (PTSG1) mLRNA, partial 124  24935 POTS7 cds Homosapiens megakaryocyte potentiating factor 126  24938 POTS7 (MPF) mRNA.Human mRNA for Apol_Human (MER5(Aop-- 127  24939 POTS7 Mouse)-likeprotein), complete cds Homo sapiens arylacetamide deacetylase (esterase)129  24942 POTS7 (AADAC) mRNA. Homo sapiens echinodermmicrotubule-associated 130  24943 POTS7 protein-like EMAP2 mRNA,complete cds Homo sapiens podocalyxin-like (PODXL) mRNA. 131  24944POTS7 Homo sapiens synaptogyrin 2 (SYNGR2) mRNA. 135  24952 POTS7 Homosapiens amyloid beta precursor protein-binding 138  24959 POTS7 protein1, 59kD (APPBP1) mRNA. Human aldose reductase mRNA, complete cds. 139 24969 POTS7 Genomic sequence from Human 9q34, complete 140  25092 POTS2sequence [Homo sapiens] Human glyceraldehyde-3-phosphate dehydrogenase141  25093 POTS2 (GAPDH) mRNA, complete cds. Homo sapiens breast cancersuppressor candidate 2 142  25098 POTS2 (bcsc-1) mRNA, complete cds Homosapiens SKB1 (S. cerevisiae) homolog (SKB1) 147  25110 POTS2 mRNA. Homosapiens prepro dipeptidyl peptidase I (DPP-I) 152  25117 POTS2 gene,complete cds Homo sapiens preferentially expressed antigen of 153  25745POTS2 melanoma (PRAME) mRNA Human translocated t(8;14) c-myc (MYC)oncogene, 154  25746 POTS2 exon 3 and complete cds Human 12S RNA inducedby poly(rI), poly(rC) and 155  25749 POTS2 Newcastle disease virus HumanmRNA for fibronectin (FN precursor) 157  25755 POTS2 Homo sapiens mRNAfor hepatocyte growth factor 159  25758 POTS2 activator inhibitor type2, complete cds Homo sapiens mRNA for KIAA0552 protein, 163  25771 POTS7complete cds Homo sapiens IMP (inosine monophosphate) 164  25775 POTS7dehydrogenase 2 (IMPDH2) mRNA Homo sapiens clone 23942 alpha enolasemRNA, 165  25787 POTS7 partial cds H. sapiens vegf gene,3′UTR 169  25799POTS7 Homo sapiens 305 ribosomal protein S7 homolog 170  25802 POTS7mRNA, complete cds Homo sapiens acetyl-Coenzyme A acetyltransferase 2172  25808 POTS7 (acetoacetyl Coenzyme A thiolase) (ACAT2) mRNA Homosapiens Norrie disease protein (NDP) mLRNA 173  25809 POTS7

[0306] Still further ovarian carcinoma polynucleotide and/or polypeptidesequences identified from the above libaries are provided below in TableVII. Sequences O574S (SEQ ID NO:183 & 185), O584S (SEQ ID NO:193) and0585S (SEQ ID NO:194) represent novel sequences. The remaining sequencesexhibited at least some homology with known genomic and/or ESTsequences. TABLE VII SEQ ID: Sequence Library 174: O565S_CRABP OS1D 175:O566S_Ceruloplasmin POTS2 176: O567S_41191.SEQ(1>487) POTS2 177:O568S_KIAA0762.seq(1>3999) POTS7 178: O569S_41220.seq(1>1069) POTS7 179:O570S_41215.seq(1>1817) POTS2 180: O571S_41213.seq(1>2382) POTS2 181:O572S_41208.seq(1>2377) POTS2 182: O573S_41177.seq(1>1370) OS1F 183:O574S_47807.seq(1>2060) n/a 184: O568S/VSGF DNA seq n/a 185:O574S_47807.seq(1>3000) n/a 186: O5685/VSGF protein seq n/a 187:449H1(57581) OS1D 188: 451E12(57582) OS1D 189:453C7_3′(57583.1)Osteonectin OS1D 190: 453C7_5(57583.2) OS1D 191:456G1_3′(57584.1)Neurotensin OS1F 192: 456G1_5′(57584.2) OS1F 193:O584S_465G5(57585) OS1F 194: O585S_469B12(57586) POTS2 195:O569S_474C3(57587) POTS7 196: 483B1_3′(24934.1)Triosephosphate POTS7197: 57885 Human preferentially POTS2 expressed antigen of melanoma 198:57886 Chromosome 22q12.1 clone POTS2 CTA-723E4 199: 57887 Homologous tomouse brain POTS2 cDNA clone MNCb-0671

[0307] Further studies on the clone of SEQ ID NO:182 (also referred toas O573S) led to the identification of multiple open reading frames thatencode the amino acid sequences of SEQ ID NO:200-202.

Example 2 Analysis of cDNA Expression Using Microarray Technology

[0308] In additional studies, sequences disclosed herein were found tobe overexpressed in specific tumor tissues as determined by microarrayanalysis. Using this approach, cDNA sequences are PCR amplified andtheir mRNA expression profiles in tumor and normal tissues are examinedusing cDNA microarray technology essentially as described (Shena et al.,1995). In brief, the clones are arrayed onto glass slides as multiplereplicas, with each location corresponding to a unique cDNA clone (asmany as 5500 clones can be arrayed on a single slide or chip). Each chipis hybridized with a pair of cDNA probes that are fluorescence-labeledwith Cy3 and Cy5 respectively. Typically, 1 μg of polyA⁺ RNA is used togenerate each cDNA probe. After hybridization, the chips are scanned andthe fluorescence intensity recorded for both Cy3 and Cy5 channels. Thereare multiple built-in quality control steps. First, the probe quality ismonitored using a panel of ubiquitously expressed genes. Secondly, thecontrol plate also can include yeast DNA fragments of whichcomplementary RNA may be spiked into the probe synthesis for measuringthe quality of the probe and the sensitivity of the analysis. Currently,the technology offers a sensitivity of 1 in 100,000 copies of mRNA.Finally, the reproducitility of this technology can be ensured byincluding duplicated control cDNA elements at different locations.

[0309] The microarray results for clones 57885 (SEQ ID NO:197), 57886(SEQ ID NO:198) and 57887 (SEQ ID NO:199) are as follows.

[0310] Clone 57885: 16/38 (42%) of ovarian tumors showed an expressionsignal value of >0.4. The mean value for all ovary tumors was 0.662 witha mean value of 0.187 for all normal tissues, which yields a 3.64 foldoverexpression level in ovary tumor relative to essential normaltissues. Normal tissue expression was elevated (>0.4) in peritoneum,skin and thymus.

[0311] Clone 57886: 16/38 (42%) of ovarian tumors showed an expressionsignal value of >0.4. The mean value for all ovary tumors was 0.574 witha mean value of 0.166 for all normal tissues which yields a 3.46 foldoverexpression level in ovary tumor relative to essential normaltissues. Normal tissue expression was elevated (>0.4) in heart, pancreasand small intestive.

[0312] Clone 57887: 17/38 (44%) of ovarian tumors showed an expressionsignal value of >0.4. The mean value for all ovary tumors is 0.744 witha mean value of 0.184 for all normal tissues which yields a 4.04 foldoverexpression level in ovary tumor relative to essential normaltissues. Normal tissue expression was elevated (>0.4) in esophagus.

Example 3 Expression of Recombinant Antigen O568S IN E. coli

[0313] This example describes the expression of recombinant antigenO568S (SEQ ID NO:177) in E. coli. This sequence was identified inExample I from the POTS 7 subtraction library using primary ovariantumor EDNA as the tracer. PCR primers specific for the open readingframe of O568S were designed and used in the specific amplification ofO568S. The PCR product was enzymatically digested with EcoRI and ligatedinto pPDM, a modified pET28 vector which had been cut with therestriction enzymes EcoRI and Eco72I. The construct sequence andorientation was confirmed through sequence analysis, the sequence ofwhich is shown in SEQ ID NO:206. The vector was then transformed intothe expression hosts, BLR (DE3) and HMS 174 (DE3) pLys S. Proteinexpression was confirmed, the sequence of which is provided in SEQ IDNO:207.

Example 4 Additional Sequence Obtained for Clone O591S

[0314] The sequence of O591S (clone identifier 57887) was used to searchpublic sequence databases. It was found that the reverse strand showedsome degree of identity to the C-terminal end of GPR39. The cDNA for thecoding region of GPR39 is disclosed in SEQ ID NO:208 and thecorresponding amino acid sequence in SEQ ID NO:209. The GPR39 codingregion contains two exons. Both O591S and GPR39, encoded by thecomplementary strand of O591S, are located on chromosome 2.

Example 5 Further Characterization of O591S and Identificaton ofExtended Sequence

[0315] O1034C is an ovary specific gene identified by electronicsubtraction. Briefly, electronic subtraction involves an analysis of ESTdatabase sequences to identify ovarian-specific genes. In the electronicsubtraction method used to indentify O1034C, sequences of EST clonesderived from ovary libraries (normal and tumor) were obtained from theGenBank public human EST database. Each ovary sequence was used as a“seed” query in a BLASTN search of the total human EST database toidentify other EST clones that share sequence with the seed sequence(clones that potentially originated from the same mRNA). EST clones withshared sequence were grouped into clusters, and clusters that sharedsequence with other clusters were grouped into superclusters. The tissuesource of each EST within each supercluster was noted, and superclusterswere ranked based on the distribution of the tissues from which the ESTsoriginated. Superclusters that comprise primarily, or solely, EST clonesfrom ovary libraries were considered to represent genes that weredifferentially expressed in ovary tissue, relative to all other normaladult tissue.

[0316] This clone was identified from the public EST databases asIntegrated Molecular Analysis of Genomics and their Expression (IMAGE)clone number 595449 (the IMAGE consortium is a repository of EST clonesand cDNA clones) and is disclosed as SEQ ID NO:210. Accession numbersAA173739 and AA173383 represents the sequence of the identified EST inGenebank. This clone is part of Unigene cluster HS.85339 (Unigene is anexperimental system for automatically partitioning Genbank sequencesinto a non-redundant set of gene-orientated clusters) and was annotatedas encoding a neurotensin-like G protein coupled receptor (GRP39).However, the inventors have discovered that IMAGE#595449 encodes a novelprotein derived from the complementary strand to that which encodes thepotential GPR39.

[0317] Microarray analysis of the clone using a series of ovary tumorspecific probes indicated that this clone was over expressed 4.95-foldin a group of ovary tumor and normal ovary samples as compared to agroup of essential normal tissue samples.

[0318] IMAGE#59449 was subjected to a Blast A search of the EST databaseand Genbank and an electronic full length clone contig (O1034C) wasgenerated by extending IMAGE#595449 and its resulting contigs tocompleteion. This process was repeated to completion when no further ESTsequences were identified to extend the consensus sequence. Thiselectronically derived clone was identified as coding a previouslydescribed clone, O591S, the sequence of which is disclosed in SEQ IDNO:211. The discovery of this ovary specific candidate is described inmore detail in Example 4.

[0319] The consensus sequence for O1034C extended further 5′ than O591Sdue to the additional sequences derived from two EST clones, accessionnumbers BF345141 and BE336607, the sequences for which are disclosed inSEQ ID NO:212 and 213 respectively. Although BF345141 diverges from theO1034C/O591S consensus at its 3′-end (possibly representing a differentsplice form), and from BE336607 at several bases at its 5′-end, the twoESTs were compared to the available matching chromosome sequence. Theywere found on human chromosome 2, clone RP11-159N20:htgs databaseaccession number AC010974. These sequences were used to extendO01034C/O591S to form a final consensus sequence for O1034C/O591S of1897 base pairs, disclosed in SEQ ID NO:214.

[0320] An open reading frame (ORF) was identified within theO1034C/O591S consensus sequence (nucleotides 260-682), the predictedtranslation of which is disclosed in SEQ ID NO:215. A BLASTx databasesearch against the Genbank database indicated that this ORF had noidentity (E value <1e-25) with any known human protein. The only matchwas with the G protein-coupled receptors, including GPR39, which theinventors have shown to be encoded at the 3′-end of O1034C/O591S on thecomplementary strand. However, the ORF did encode a protein that had 93%similarity (131/141 amino acids) and 91% identity (129/141 amino acids)with an un-named murine product (Accession #BAA95101), suggesting thatthis is a real translation product that represents a novel humanovary-specific antigen.

[0321] The novelty of O1034C/O591S was confirmed by Northern Blotanalysis using single stranded probes that complement either GRP39 orO1034C/O591S. The strand-specific O1034C/O591S probe specificallyhybridized to the ovary tumor samples probed on the Northen blot, whilstall samples were negative when probed with GPR39. In addition real-timePCR was performed using primers specific for either GPR39 orO1034C/O591S. These results further demonstrated the differentialexpression profiles of the two sequences. This protein is a putativemembrane protein as determined from Corixa's Tmpred protein predictionalgorithm.

Example 6 Expression Analysis and Further Characterization of OvarianSequence O568S

[0322] The ovarian sequence O568S was originally identified as cDNAclone 24742 (SEQ ID NO:118). Using clone 24742 as a query sequence tosearch public sequence databases, the sequence was found to have a highdegree of homology with KIAA0762 (SEQ ID NO:177) and with VSGF. The DNAsequence for VSGF is provided in SEQ ID 184 and the VSGF proteinsequence is provided in SEQ ID NO:186.

[0323] Real-time PCR (see Gibson et al., Genome Research 6:995-1001,1996; Heid et al., Genome Research 6:986-994, 1996) is a technique thatevaluates the level of PCR product accumulation during amplification.This technique permits quantitative evaluation of mRNA levels inmultiple samples. Briefly, mRNA is extracted from tumor and normaltissue and cDNA is prepared using standard techniques. Real-time PCR isperformed, for example, using a Perkin Elmer/Applied Biosystems (FosterCity, Calif.) 7700 Prism instrument. Matching primers and fluorescentprobes are designed for genes of interest using, for example, the primerexpress program provided by Perkin Elmer/Applied Biosystems (FosterCity, Calif.). Optimal concentrations of primers and probes areinitially determined by those of ordinary skill in the art, and control(e.g., β-actin) primers and probes are obtained commercially from, forexample, Perkin Elmer/Applied Biosystems (Foster City, Calif.). Toquantitate the amount of specific RNA in a sample, a standard curve isgenerated using a plasmid containing the gene of interest. Standardcurves are generated using the Ct values determined in the real-timePCR, which are related to the initial cDNA concentration used in theassay. Standard dilutions ranging from 10-10⁶ copies of the gene ofinterest are generally sufficient. In addition, a standard curve isgenerated for the control sequence. This permits standardization ofinitial RNA content of a tissue sample to the amount of control forcomparison purposes.

[0324] By RealTime PCR analysis, 0568 was highly overexpressed in themajority of ovary tumors and ovary tumor metastases tested relative tonormal ovary tissue and relative to an extensive normal tissue panel.Little or no expression was observed in normal esophagus, spinal cord,bladder, colon, liver, PBMC (activated or resting), lung, skin, smallintestine, stomach, skeletal muscle, pancreas, dendritic cells, heart,spleen bone marrow, thyroid, trachea, thymus, bronchia, cerebellum,ureter, uterus and peritoneum epithelium. Some low level expression wasobserved in normal breast, brain, bone, kidney, adrenal gland andsalivary gland, but the expression levels in these normal tissues weregenerally at least several fold less than the levels observed in ovarytumors overexpressing O568S.

[0325] Moreover, a series of Northern blots was performed which alsodemonstrated that the ORF region of 0568S is specifically overexpressedin ovary tumors. The initial blot contained RNA from a series of normaltissues as well as from ovary tumors. This blot was probed using, as alabeled probe, DNA from 0568s that corresponded to the 3′UTR of the VSGFsequence disclosed in SEQ ID NO:184. This blot revealed an ovarytumor-specific 5.0 Kb message as well as a potential 3.5 Kb brainspecific message and a ubiquitously expressed 1.35 Kb message.

[0326] Another Northern blot was performed with RNAs from a number ofdifferent brain tissues and probed with the 3′UTR region as above. Fiveof eleven brain samples showed overexpression of the 3.5Kb message. Inorder to determine whether the ORF region of O568S was specificallyoverexpressed in ovary tumors, a series of three blots was carried outusing three separate probes designed from within the VSGF ORF of O568S.Results from these experiments clearly indicated that only the 5.0 Kbmessage is expressed in ovary tumor.

Example 7 Synthesis of Polypeptides

[0327] Polypeptides are synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence is attached to the amino terminus ofthe peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support is carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides are precipitated in cold methyl-t-butyl-ether.The peptide pellets are then dissolved in water containing 0.1%trifluoroacetic acid (TFA) and lyophilized prior to purification by C18reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1%TFA) in water (containing 0.1% TFA) is used to elute the peptides.Following lyophilization of the pure fractions, the peptides arecharacterized using electrospray or other types of mass spectrometry andby amino acid analysis.

[0328] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1 215 1 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C or G 1caacctcact agtaaatgaa agaaatattg taatttgtat ttgatctgct gggtctttgg 60agtcagaact ggttttatca gcagtttgat cttctgaggt ctggtatgta gtttgctggc 120ccacagaacc ttcacgtgta ttcacagcct caatgccata aggaaactct tttagaagtt 180ctgacagctg gtcatgtagg tataagacag gtgccttatc actgtggatt tcatttcttg 240caggatcttg gggagtatag ttgctggatg catctatttc ctgagggtaa atatcctcct 300ggncgacgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 360tgccttctan ttgccancca tntgttgttt gcccct 396 2 396 DNA Homo sapien 2cgaccaaaaa gtaaactcca agtgaacatc aaatcaaatc taatcctttt ggccacatga 60ctggttgttc tttatctcat agttacaatg aatcatataa actgtagact gccactacca 120cgatacttct gtgacacaga aggaatgtcc tatttgccta tctatctgag gaatgttaaa 180tagagaaaaa tagattataa aacaacctgg aggtcacagg attctgagat aatccctctg 240ttaaaaaaca tctgaacagc aaatgtccaa tctgtaataa aatagttaaa ggtccaagtc 300aagtccactt ctacttggct ggcccagcac aagaaatcta acagcacttt gtaatcattt 360tgcttttcta attttcccgg aggacatggg ccattg 396 3 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 3 cgcccttttt tttttttttttnattggnnn aantcncttt nantnnaaaa acntgnangg 60 naancccann cccnnggnaccannnccagg agttgggtgg anactgagtg gggtttgtgt 120 gggtgagggg gcatctactcctnttgcaac aagccaaaag tagaacagcc taaggaaaag 180 tgacctgcct tggagccttagtccctccct tagggccccc tcagcctacc ctatccaagt 240 ctgaggctat ggaagtctccctcctagttc actagcaggt tccccatctt ttccaggctg 300 cccctagcac tccacgtttttctgaaaaaa tctanacagg ccctttttgg gtacctaaaa 360 cccagctgag gttgtgagcttgtaaggtaa agcaag 396 4 396 DNA Homo sapien misc_feature (1)...(396) n =A,T,C or G 4 gaccaatcct tgncncacta ncaaaangac cccnctnacc nccaggaactgaacctnnnt 60 gtnnacctcc nnctgcnnag ccntatntcc aanatcaccc accgtatccactgggaatct 120 gccagcctcc tgcgatcaga agagaccaat cgaaaatgag ggtttcacantcacagctga 180 aggaaaaggc caaggcacct tgtcggnggn gacaatgtac catgctaaggccaaagatca 240 actcacctgt aataaattcg acctcaaggt caccataaaa ccagcaccggaacagaaaaa 300 gaggcctnag gatgcccaag aaacactttt gatcctttga aaactgtaccaaggtaccgg 360 ggggagaccc aggaaaggnc cnttatgtnt nnntnt 396 5 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 5 gacgccggagctgccgcgcc agtcgcctag caggtcctct accggcttat tcctgtgccg 60 gatcttcatcggcacagggg ccactgagac gtttctgcct ccctctttct tcctccgctc 120 tttctcttccctctngttta gtttgcctgg gagcttgaaa ggagaaagca cnggggtcgc 180 cccaaaccctttctgcttct gcccatcaca agtgccacta ccgccatggg cctcactatc 240 tcctccctcttctcccgact atttggcaag aagcagatgc gcattttgat ggttggattg 300 gatgctgctggcaagacaac cattcttgat aaactgaaag tanggganat aagnaccacc 360 atttctaccattgggtttaa tgggggaaac agtana 396 6 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 6 acgggaggcg ccgggaagtc gacggcgccg gcggctcctgcaggaggcca ctgtctgcag 60 ctcccgtgaa gatgtccact ccagacccac ccctgggcggaactcctcgg ccaggtcctt 120 ccccgggccc tgcccttccc ctggagccat gctgggccctagcccgggtc cctcgccggg 180 ctccgcccac agcatgatgg ggcccagccc angggccgccctcagcagga caccccatcc 240 ccacccaggg gcctggaggg taccctcagg acaacatgcaccagatgcac aagcccatgg 300 agtccatgca tgagaagggc atgtcggacg acccgcgctacaaccagatg aaaggaatgg 360 ggatgcggtc agggggccat gctgggatgg ggcccc 396 7396 DNA Homo sapien 7 acccgagagt cgtcggggtt tcctgcttca acagtgcttggacggaaccc ggcgctcgtt 60 ccccaccccg gccggccgcc catagccagc cctccgtcacctcttcaccg caccctcgga 120 ctgccccaag gcccccgccg ccgctccagc gccgcgcagccaccgccgcc gccgccgcct 180 ctccttagtc gccgccatga cgaccgcgtc cacctcgcaggtgcgccaga actaccacca 240 ggactcagag gccgccatca accgccagat caacctggagctctacgcct cctacgttta 300 cctgtccatg tcttactact ttgaccgcga tgatgtggctttgaagaact ttgccaaata 360 ctttcttcac caatctcatg aggagaggga acatgc 396 8396 DNA Homo sapien 8 cgacaacaag gttaatacct tagttcttaa cattttttttctttatgtgt agtgttttca 60 tgctaccttg gtaggaaact tatttacaaa ccatattaaaaggctaattt aaatataaat 120 aatataaagt gctctgaata aagcagaaat atattacagttcattccaca gaaagcatcc 180 aaaccaccca aatgaccaag gcatatatag tatttggaggaatcaggggt ttggaaggag 240 tagggaggag aatgaaggaa aatgcaacca gcatgattatagtgtgttca tttagataaa 300 agtagaaggc acaggagagg tagcaaaggc caggcttttctttggttttc ttcaaacata 360 ggtgaaaaaa acactgccat tcacaagtca aggaac 396 9396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C or G 9 tcgacatcgcggcaactttt tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc 60 agtgctaccagtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg 120 tgaattgcacggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg 180 ggatcatgtaccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt 240 accagtccttctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc 300 ctctttgtaacgggccaagg nccaaaaaaa ggggaaagtt ctgncctcgg ccctcaggcc 360 agggctccgcaccaccatcc tgttcctcaa attagc 396 10 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 10 cctttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttttttttttttt ttttaaaaaa aaaannnttt 120 tttttttttn aaaaaaangg gnnnnnttttttncccnnnn gggngggggg ggggnnnnnt 180 ttnaaanaaa aaaaccnnaa annnnnggggnnnannnaan nncccncccc naancnntaa 240 aaaannnggn aaaanagggg gggnannnnnnnggggggna aaantttttt ttttttnaag 300 ggnnnggnaa aaaantnnnn nnntttttttttnnaanngg gnnaaaaaaa aaaaaaaaaa 360 attttttngg gntnaggggn ngggggaaaancccna 396 11 396 DNA Homo sapien 11 agaacacagg tgtcgtgaaa actacccctaaaagccaaaa tgggaaagga aaagactcat 60 atcaacattg tcgtcattgg acacgtagattcgggcaagt ccaccactac tggccatctg 120 atctataaat gcggtggcat cgacaaaagaaccattgaaa aatttgagaa ggaggctgct 180 gagatgggaa agggctcctt caagtatgcctgggtcttgg ataaactgaa agctgagcgt 240 gaacgtggta tcaccattga tatctccttgtggaaatttg agaccagcaa gtactatgtg 300 actatcattg atgccccagg acacagagactttatcaaaa acatgattac agggacatct 360 caggctgact gtgctgtcct gattgttgctgctggt 396 12 396 DNA Homo sapien 12 cgaaaacctt taaaccccgg tcatccggacatcccaacgc atgctcctgg agctcacagc 60 cttctgtggt gtcatttctg aaacaagggcgtggatccct caaccaagaa gaatgtttat 120 gtcttcaagt gacctgtact gcttggggactattggagaa aataaggtgg agtcctactt 180 gtttaaaaaa tatgtatcta agaatgttctagggcactct gggaacctat aaaggcaggt 240 atttcgggcc ctcctcttca ggaatcttcctgaagacatg gcccagtcga aggcccagga 300 tggcttttgc tgcggccccg tggggtaggagggacagaga gacagggaga gtcagcctcc 360 acattcagag gcatcacaag taatggcacaattctt 396 13 396 DNA Homo sapien 13 accacaggct ggccacaaga agcgctggagtgtgctggcg gctgcaggcc tacggggcct 60 ggtccggctg ctgcacgtgc gtgccggcttctgctgcggg gtcatccgag cccacaagaa 120 ggccatcgcc accctgtgct tcagccccgcccacgagacc catctcttca cggcctccta 180 tgacaagcgg atcatcctct gggacatcggggtgcccaac caggactacg aattccaggc 240 cagccagctg ctcacactgg acaccacctctatccccctg cgcctctgcc ctgtcgcctc 300 ctgcccggac gcccgcctgc tggccggctgcgagggcggc tgctgctgct gggacgtgcg 360 gctggaccag ccccaaaaga ggagggtgtgtgaagt 396 14 396 DNA Homo sapien 14 acggcgtcct cgtggaagtg acatcgtctttaaaccctgc gtggcaatcc ctgacgcacc 60 gccgtgatgc ccagggaaga cagggcgacctggaagtcca actacttcct taagatcatc 120 caactattgg atgattatcc gaaatgtttcattgtgggag cagacaatgt gggctccaag 180 cagatgcagc agatccgcat gtcccttcgcgggaaggctg tggtgctgat gggcaagaac 240 accatgatgc gcaaggccat ccgagggcacctggaaaaca acccagctct ggagaaactg 300 ctgcctcata tccgggggaa tgtgggctttgtgttcacca aggaggacct cactgagatc 360 agggacatgt tgctggccaa taaggtgccagctgct 396 15 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 15 accgcgcggg cacagggtgc cgctgaccga ggcgtgcaaa gactccagaa ttggaggcat60 gatgaagact ctgctgctgt ttgtggggct gctgctgacc tgggagagtg ggcaggtcct 120gggggaccag acggtctcag acaatgagct ccaggaaatg tccaatcagg gaagtaagta 180cgtcaataag gaaattcaaa atgcttgtca acggggtgaa acagataaag actctcatag 240aaaaaacaaa cgaagagcgc aagacactgc tcagcaacct agaagaagcc aagaagaaga 300aagaggatgc cctaaatgag accagggaat canagacaaa gctgaaggag ctcccaggag 360tgtgcaatga gaccatgatg gccctctggg aagagt 396 16 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 16 tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttngggggg 120 nnnaaanttt tttntnanannnnngggnaa aaaaaaaaaa aanaangggg gnnntnnggc 180 ccnnnanaaa aaaanngnnaannaancccc ccnnnnnnnc ccncnnntnn ggaaananna 240 aaaccccccc cngggnngggnnaaaaannc ccnggggnan tttttatnnn annccccccc 300 ccnggggggg gnggaaaaaaaaaantnccc ccnannaaaa nnggggnccc cccnttttnc 360 aaaanggggg nccgggccccccnnantntt nggggg 396 17 396 DNA Homo sapien 17 accacactaa ccatataccaatgatggcgc gatgtaacac gagaaagcac ataccaaggc 60 caccacacac cacctgtccaaaaaggcctt cgatacggga taatcctatt tattacctca 120 gaagtttttt tcttcgcaggatttttctga gccttttacc actccagcct agcccctacc 180 ccccaactag gagggcactggcccccaaca ggcatcaccc cgctaaatcc cctagaagtc 240 ccactcctaa acacatccgtattactcgca tcaggagtat caatcacctg agctcaccat 300 agtctaatag aaaacaaccgaaaccaaata attcaagcac tgcttattac aattttactg 360 ggtctctatt ttaccctcctacaagcctca gagtac 396 18 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 18 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttantcnaaaggg 60 gaaggnccct ttttattaaa nttggncatt ttactttnct tttttnaaaangctaanaaa 120 aaanttttnt ttntncttaa aaaaaccctn natntcacna ncaaaaaaaacnattcccnc 180 ntncnttttg tgataaaaaa aaaggcaatg gaattcaacn tancctaanaaaactttncc 240 tgggaggaaa aaaaattnnt ccgngggaaa cacttggggc tntccaaantgnanccatnc 300 tangaggacc ntctntaaga tttccaaang aaaccccttc ctnccaaangnantaccccg 360 ntgcctacnn cccataaaaa aaacctcanc cntaan 396 19 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 19 tttttttttttttttttttt tttttttttt tttttttttt ttttttntgg tctgggcttt 60 tattttacnaaaaanctaan ggnaaanntn cnttaaacta antngaanac aaagtnttaa 120 ngaaaaaggnctgggggnnt cntttacaaa aanggncngg gncanntttg ggcttaaaan 180 ttcaaaaagggnncntcaaa ngggtttgca tttgcatgtt tcancnctaa ancgnangaa 240 naaacccnggngnccnctgg gaaaagttnt tnanctncca aaanatnaan tntttgnanc 300 agggnntttttgggnaaaaa aannanttcc anaaactttc catcccctgg ntttgggttc 360 ggccttgngttttcggnatn atntccntta angggg 396 20 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 20 tttttttttt tttttttttt ttttttctnaacaaaccctg ttnttgggng ggngngggta 60 taatactaag ttganatgat ntcatttacgggggaaggcn ctttgtgaan naggccttat 120 ttctnttgnc ctttcgtaca gggaggaatttgaagtaaan anaaaccnac ctggattact 180 ccggtctgaa ctcaaatcac gtaggactttaatcgttgaa caaacaaacc tttaatagcg 240 gctgcnccat tgggatgtcc tgatccaacatcgaggncgt aaaccctatt gttgatatgg 300 actctaaaaa taggattgcg ctgttatccctagggtaact tgttcccgtg gtcaaagtta 360 ttggatcaat tgagtataag tagttcgctttgactg 396 21 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 21 acatanatnt tatactanca ttnaccatct cacttgnagg aanactanta tatcnctcac60 acctnatatc ctncntacta tgcctagaag gaataatact atngctgttn attatancta 120ctntnataac cctnaacacc cactccctct tanccaatat tgtgcctatt gccatactag 180tntttgccgc ctgcnaagca gnggngggcc tanccntact agnctcaatc tccaacacnt 240atggcctana ctacgtacat aacctaaacc tactcnaatg ctaaaactaa tcnncccaac 300anttatntta ctaccactga catgactttc caaaaaacac atantttgaa tcaacncanc 360cacccacanc ctanttatta ncatcatccc cntact 396 22 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 22 tttttttttt ttttganaaaagccggcata aagcactttt attgcaataa taaaacttga 60 gactcataaa tggtgctgggggaagggtgc agcaacgatt tctcaccaaa tcactacaca 120 ggacagcaaa ggggtgagaaggggctgagg gaggaaaagc caggaaactg agatcagcag 180 agggagccaa gcatcaaaaaacaggagatg ctgaagctgc gatgaccagc atcattttct 240 taanagaaca ttcaaggatttgtcatgatg gctgggcttt cactgggtgt taagtctaca 300 aacagcacct tcaattgaaactgtcaatta aagttcttaa gatttaggaa gtggtggagc 360 ttggaaagtt atgagattacaaaattcctg aaagtc 396 23 396 DNA Homo sapien 23 acaaaggcgg ttccaagctaaggaattcca tcagtgcttt tttcgcagcc accaaattta 60 gcaggcctgt gaggttttcatatcctgaag agatgtattt taaagctttt tttttttaat 120 gaaaaaatgt cagacacacacaaaagtaga atagtaccat ggagtcccca cgtacccagc 180 ctgcagcttc aacagttaccacatttgcca accggagaga ctgccaaggc aggaaaaagc 240 cctggaaagc ccacggcccctttttccctt gggtcagagg ccttagagct ggctgccaaa 300 gcagccaacc aaaggggcagctcagctcct tcgtggcacc agcagtgttc ctgatgcagt 360 tgaagagttg atgtctttgacaacatacgg acactg 396 24 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 24 cgactatcct ctcagattct tatctggcac taatttataa ctattatattatcagagact 60 atgtagcaat atatcagtgc acaggcgcat cccaggcctg tacagatgtatgtctacacg 120 taagtataaa tgaatttgca taccaggttt tacacttgca tctctaatagagattaaaaa 180 caacaaattg gcctcttcct aagtatatta atatcattta tccttacattttatgcctcc 240 ccctaaatta atgactgagt tggtggaaag cggctaggtt ttattcatactgttttttgt 300 tctcaacttc aanagtaatc tacctctgaa aaatttntan tttaatattnnnnnnnagga 360 atttgngcca ctttannnct tncnntntnn tnnccn 396 25 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 25 tttttttttttttttttttt gtcttttaaa aaatataaaa gtgttattat tttaaaacat 60 caagcattacagactgtaaa atcaattaan aactttctgt atatgaggac aaaaatacat 120 ttaanacatatacaanaaga tgctttttcc tgagtagaat gcaaactttt atattaagct 180 tctttgaattttcaaaatgt aaaataccaa ggctttttca catcagacaa aaatcaggaa 240 tgttcaccttcacatccaaa aagaaaaaaa aaaaaaancc aattttcaag ttgaagttna 300 ncaanaatgatgtaaaatct gaaaaaagtg gccaaaattt taanttncaa canannngnn 360 ncagntttnatggatctntn nnnnnncttc nnntnn 396 26 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 26 gacgctcccc cctccccccg agcgccgctccggctgcacc gcgctcgctc cgagtttcag 60 gctcgtgcta agctagcgcc gtcgtcgtctcccttcagtc gccatcatga ttatctaccg 120 ggacctcatc agccacgatg agatgttctccgacatctac aagatccggg agatcgcgga 180 cgggttgtgc ctggaggtgg aggggaagatggtcagtagg acagaaggta acattgatga 240 ctcgctcatt ggtggaaatg cctccgctgaaggccccgag ggcgaaggta cccgaaagca 300 cagtaatcac tgnngncnat nttgtcatgaaccatcacct gcnngaaaca annttnacaa 360 aanaancctn cnnnnannnc ctnnnnnattncnnnn 396 27 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 27 tttttttttt tttttttttt tttttttttt tttttttttt tggctaaant ttatgtatac60 nggttnttca aangnggggg aggggggggg gcatccatnt anncncncca ggtttatggn 120gggntnttnt actattanna nttttcnctt caaancnaag gnttntcaaa tcatnaaaat 180tattaanatt ncngctgnta aaaaaangaa tgaaccnncn nanganagga nntttcatgg 240ggggnatgca tcggggnann ccnaanaacc ncggggccat tcccganagg cccaaaaaat 300gtttnnnnaa aaagggtaaa nttacccccn tnaantttat annnnaaann nnannnnagc 360ccaannnttn nnnnnnnnnn nnnccnnnna nnnnnn 396 28 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 28 cgaccttttt ttttttttttatagatgaaa gagggtttat ttattaatat atgatagcct 60 tggctcaaaa aagacaaatgagggctcaaa aaggaattac agtaacttta aaaaatatat 120 taaacatatc caagatcctaaatatattat tctccccaaa agctagctgc ttccaaactt 180 gatttgatat tttgcatgttttccctacgt tgcttggtaa atatatttgc ttctcctttc 240 tgcaatcgac gtctgacagctgatttttgc tgttttgnca acntgacgtt tcaccttntg 300 tttcaccant tctggaggaattgttnaaca ncttacanca ctgccttgaa naaannnnan 360 gcctcaaaag ntcttgnnctatnctnnttc ntnnnt 396 29 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 29 gacttgctca tttagagttt gcaggaggct ccatactagg ttcagtctgaaagaaatctc 60 ctaatggtgc tatagagagg gaggtaacag aaagactctt ttagggcatttttctgactc 120 atgaaaagag cacagaaaag gatgtttggc aatttgtctt ttaagtcttaaccttgctaa 180 tgtgaatact gggaaagtga tttttttctc actcgttttt gttgctccattgtaaagggc 240 ggaggtcagt cttagtggcc ttgagagttg cttttggcat ttaaatattctaagagaatt 300 aactgtattt cctgtcacct attcactant gcangaaata tacttgctccaaataagtca 360 ntatgagaag tcactgtcaa tgaaanttgn tttgtt 396 30 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 30 tttttttttttttttttttg aaatttanaa acaaatttta tttaagatct gaaatacaat 60 tcctaaaatatcaacttttc canaaaaccg tggctacaca ataatgcatt gcctctatca 120 tgttanaacgtgcattanac tcaaatacaa aaaccatgaa acaaatcacc atccttcaac 180 aatttgagcaaagatagaat gcctaagaac aacatagatg gacttgcaga ggatgggctg 240 ttttacttcaagcnccataa aaaaaaaaaa gagcncaaat gcattgggtt ttcaggtnta 300 tacattaagnngaacctttg gcactaggaa tcagggcgtt ttgtcacata gcnttaacac 360 atnttaaaaaattntgtant gtcaaaggga tangaa 396 31 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 31 gacgggccag ggccatctgg aaagggaactcggcttttcc agaacgtggt ggatcatctg 60 tcgggtgtgt ggtgaacacg ttcagttcatcagggcctac gctccgggaa ggggccccca 120 gctgtggctc tgccatgccg ggctgtgtttgcagctgtcc gagtctccat ccgcctttag 180 aaaaccagcc acttcttttc ataagcactgacagggccca gcccacagcc acaggtgcga 240 tcagtgcctc acgcaggcaa atgcactgaaacccaggggc acacncncgc agagtgaaca 300 gtgagttccc ccgacagccc acgacagccaggactgccct ccccaccccn ccccgacccc 360 angancacgg cacacanntc ancctctnanctngct 396 32 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 32 cgactggcct cataccttgt ctacacagtc cctgcacagg gttcctaacc tgtggttagt60 aaagaatgtc actttctaac aggtctggaa gctccgagtt tatcttggga actcaagagg 120agaggatcac ccagttcaca ggtatttgag gatacaaacc cattgctggg ctcggcttta 180aaagtcttat ctgaaattcc ttgtgaaaca gagtttcatc aaagccaatc caaaaggcct 240atgtaaaaat aaccattctt gctgcacttt atgcaaataa tcaggccaaa tataagacta 300cagtttattt acaatttgtt tttaccaaaa atgaggacta nagagaaaaa tggtgctcca 360aagcttatca tacatttgtc attaagtcct agtctc 396 33 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 33 cctttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt 120 nngnnntntn nnnnannaaaaaaaaaaaaa aannnnnnna aaaaaaannn nnnnnnnnnt 180 tttnnggggg gnttttnanngnannttnnn nttnnnnnaa anccccnnng ggnngggggg 240 nntnnnnnng gnaaaaaaannnnnnggggn cnnnngggnc cncncccnan nnnnaaaann 300 nnnggntttt ttnnttttnaaaaaaanngn nnnnnaacaa aantttttnn nnaanttttn 360 gggggaaann ncccntttntttttttnnan nnnnnn 396 34 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 34 acggaccnag ctggaggagc tgggtgtggg gtgcgttggg ctggtggggaggcctagttn 60 gggtgcaagt angtctgatt gagcttgtgt tgtgctgaag ggacagccctgggtctaggg 120 ganagagncc ctgagtgtga gacccacctt ccccngtccc agcccctcccanttccccca 180 gggacggcca cttcctgntc cccgacncaa ccatggctga agaacaaccgcaggtcgaat 240 tgttcntgaa ggctggcagt gatggggcca agattgggaa ctgcccattctcccacagac 300 tgttnatggt actgtggctc aaggnagtca ccttcaatgt taccaccnntgacaccaaaa 360 ggcggaccna nacagtgcan aagctgtgcc canngg 396 35 396 DNAHomo sapien 35 tcgaccaaaa tcaaatctgg cactcacaag ccctggccga cccccaatgggttttaccac 60 tccccctcta gaccctgtct tgcaaaatcc tctccctagc cagctagtattttctgggct 120 aaagactgta caaccagttc ctccatttta tagaagttta ctcactccaggggaaatggt 180 gagtcctcca acctcccttt caaccagtcc catcattcca accagtggtaccatagagca 240 gcaccccccg ccaccctctg agccagtagt gccagcagtg atgatggccacccatgagcc 300 cagtgctgac ctggcaccca agaaaaagcc caggaagtca agcatgcctgtgaagattga 360 gaaggaaatt attgataccg ccgatgagtt tgatga 396 36 396 DNAHomo sapien 36 tcgacgggaa gagcctgcta cggtggactg tgagactcag tgcactgtcctcctcccagc 60 gaccccacgc tggaccccct gccggaccct ccacccttcg gcccccaagcttcccagggg 120 cttcctttgg actggactgt ccctgctcat ccattctcct gccacccccagacctcctca 180 gctccaggtt gccacctcct ctcgccagag tgatgaggtc ccggcttctgctctccgtgg 240 cccatctgcc cacaattcgg gagaccacgg aggagatgct gcttgggggtcctggacagg 300 agcccccacc ctctcctagc ctggatgact acgtgaggtc tatatctcgactggcacagc 360 ccacctctgt gctggacaag gccacggccc agggcc 396 37 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 37 cgacggtgtcagcaactggc catgccacag cacataaaga ttacagtgac aagaaaaaca 60 ttgtttgaggattcctttca acagataatg agcttcagtc cccaagatct gcgaagacgt 120 ttgtgggtgatttttccagg agaagaaggt ttagattatg gaggtgtagc aagagaatgg 180 ttctttcttttgtcacatga agtgttgaac ccaatgtatt gcctgtttga atatgcaggg 240 aaggataactactgcttgca gataaacccc gcttcttaca tcaatccaga tcacctgaaa 300 tattttcgttttattggcag atttattgcc atggctctgt tccatgggaa aattcataga 360 cacgggtttttctttnccat tctataagcg tatctt 396 38 396 DNA Homo sapien 38 cgaccaaaatgataaatagc tttaagaatg tgctaatgat aaatgattac atgtcaattt 60 aatgtacttaatgtttaata ccttatttga ataattacct gaagaatata ttttttagta 120 ctgcatttcattgattctaa gttgcacttt ttacccccat actgttaaca tatctgaaat 180 cagaatgtgtcttacaatca gtgatcgttt aacattgtga caaagtttaa tggacagttt 240 tttcccatatgtatatataa aataatgtgt tttacaatca gtggcttaga ttcagtgaaa 300 tacagtaattcattcaatta tgatagtatc tttacagaca ttttaaaaat aagttatttt 360 tatatgctaatattctatgt tcaagtggaa tttgga 396 39 396 DNA Homo sapien 39 tcgaccaagaatagatgctg actgtactcc tcccaggcgc cccttccccc tccaatccca 60 ccaaccctcagagccacccc taaagagata ctttgatatt ttcaacgcag ccctgctttg 120 ggctgccctggtgctgccac acttcaggct cttctccttt cacaaccttc tgtggctcac 180 agaacccttggagccaatgg agactgtctc aagagggcac tggtggcccg acagcctggc 240 acagggcaagtgggacaggg catggccagg tggccactcc agacccctgg cttttcactg 300 ctggctgccttagaaccttt cttacattag cagtttgctt tgtatgcact ttgttttttt 360 ctttgggtcttgtttttttt ttccacttag aaattg 396 40 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 40 tttttttttt ttttgttatt tagtttttatttcataatca taaacttaac tctgcaatcc 60 agctaggcat gggagggaac aaggaaaacatggaacccaa agggaactgc agcgagagca 120 caaagattct aggatactgc gagcaaatggggtggagggg tgctctcctg agctacagaa 180 ggaatgatct ggtggttaan ataaaacacaagtcaaactt attcgagttg tccacagtca 240 gcaatggtga tcttcttgct ggtcttgccattcctggacc caaagcgctc catggcctcc 300 acaatattca tgccttcttt cactttgccaaacaccacat gcttgccatc caaccactca 360 gtcttggcag tgcanatgaa aaactgggaaccattt 396 41 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 41 tcgacctctt gtgtagtcac ttctgattct gacaatcaat caatcaatgg cctagagcac60 tgactgttaa cacaaacgtc actagcaaag tagcaacagc tttaagtcta aatacaaagc 120tgttctgtgt gagaattttt taaaaggcta cttgtataat aacccttgtc atttttaatg 180tacaaaacgc tattaagtgg cttagaattt gaacatttgt ggtctttatt tactttgctt 240cgtgtgtggg caaagcaaca tcttccctaa atatatatta cccaaagnaa aagcaagaag 300ccagattagg tttttgacaa aacaaacagg ccaaaagggg gctgacctgg agcagagcat 360ggtgagaggc aaggcatgag agggcaagtt tgttgt 396 42 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 42 cttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt 60 aaaanccnna nnaanananggnaannnann aaaaaannca aaccncntnt anaaaangcc 120 nntntnaggg ggggggttcaaaaccaaang gnngntngga ngnaaannna aaanttnnnn 180 gggggnanaa anaaaaagggnngaaanntg acccnanaan gaccngaaan cccgggaaac 240 cnngggntan aaaaaaagntganccctaaa nncccccgna aaanggggga agggnaannc 300 caaatccnnt gngggttgggggnggggaaa aaaaaaaccc cnaaaaantg naaaaaaccg 360 ggnttnaaan atttgggttcgggggntttn tnttaa 396 43 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 43 tttttttttt ttttgcttca ctgctttatt tttgaaatca caagcaattcaaagtgatca 60 tcattgaggc ttctgttaaa agttcttcca aagttgccca gttttaanattaaacaatat 120 tgcactttaa gatgaactaa cttttgggat tctcttcaaa gaaggaaagtattgctccat 180 ctgtgctttt cttanactaa aagcatactg canaaaactc tattttaaaaatcaacactg 240 cagggtacag taacatagta aagtacctgc ctattttana atcctanagaacatttcatt 300 gtaagaaact agcccattat ttaagtgtcc acagtatttt tcatttcantggtccaagat 360 gccaaggttt ccaaacacaa tcttgttctc taatac 396 44 396 DNAHomo sapien 44 gacctagttt tacctcttaa atatctctgt tcccttctaa gttgtttgctgtgttttctt 60 cagagcaaga aggttatatt ttttaaaatt tacttagtaa tgcacattcaaaacacacat 120 caagtcttca ggataaagtt caaaaccgct gtcatggccc catgtgatctctccctcccc 180 tacccctcta tcatttagtt tcttctgcgc aagccactct ggcttcctttcagttttgtg 240 gttcccgttt ttagctagtt cagtggtttt caatgggcat ttcttgcctttttttttcta 300 aacgacaaat agaaatacat cttctttatt atcctccaaa tccaattcagaggtaatatg 360 ctccacctac acacaatttt agaaataaat taaaaa 396 45 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 45 ttttttttttttttaaannt tntaaatttt taatgaaann ganttagaac aatgtattat 60 tnacatgtaaataaaaaaag agancataan ccccatatnc tcnnnaaagg aaggganacn 120 gcnggccntttatnagaana nnnnncatat aagaccccat taagaagaat ctggatctaa 180 anacttncaaacaggagttc acagtangtg aacagcannc cctaatccca ctgatgtgat 240 gnttcanataaaatcancan cgntgatcgg gnatcnnanc aatntgancg gaanannact 300 gctcnatatntttnaggann cngatgtggt cattttttac aaagataatg gccacaccct 360 tccngnccgaatcgancnga nctcccnntt ctgtgn 396 46 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 46 tttttttttt tttttttttc tganacagagtctcattctg ttgcctaggc tggattgcag 60 tggtgccatc tcggctcact gcaacctccgcctcctgggt tccanaaatt ctcctgcctc 120 agcctcccgg gtagctggga ctanaggcacacgccaccac gccaggctaa tttttatatt 180 tttagtanan atggcgtttc accatgttgaccanactgat ctcgaactcc cgacctcgtg 240 atccacccac ctcggcctcc caaagtgctgggattacagg cgtgaaacca ccaggcccgg 300 cctgaaatat ctatttnttt tcagattatttttaaaattc catttgatga atcttttaaa 360 gtgagctana naaagtgngt gtgtacatgcacacac 396 47 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 47 tttttttttt tttttttgct gttgccaact gtttattcag ggccctgaac gggtggtgcg60 tggacatgca acacactcgg gcccacagca gcgtgaccgg ccgctcccaa gccccgggcg 120cacaaccaca gccaggagca gcccctgcca ccactgggcc accgtccagg gccccacagg 180accagccgaa ggtgccccgg gccgaggcca gctgggtcag gtgtacccct agcctggggt 240tgagtgagga gcggcacccc cagtatcctg tgtaccccaa gttgcccagn aggccgaggg 300ggccttgggc tccatctgca ctggccaccc cgtgccaagc atcacagctg cgtgagcagg 360tttgtgtgtg agcgtgtggc ggggcctggt tgtccc 396 48 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 48 ctgggcctgt gccgaagggtctgggcagat cttccaaaga tgtacaaaat gtagaaattg 60 ccctcaagca aatgcaaagatgctcaacac ccttagtcat caagaaaatg caaatggaat 120 ccacagagag atactgcacactgacaaaga tggtcgtatt actaaaggtg aataaccagc 180 gcggggggca cgtggagtcactggaacatt tgtgcaatgc tggtgggaat gtcaacccgt 240 gcggccctct ggaataagcctggcagctcc tccaagagtt acccgtgtga cccagcaatt 300 ccactcctag ctccacccacaggaattgaa agcaaagacg caaacagatg cctgtgcacc 360 aaagttcacg gcagcatccttcgccatagt ggnaan 396 49 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 49 accccaaaat gggaaaggaa aagactcata tnaacattgn cgtnattggacacgtacatt 60 cggncaagtn caccactact ggncatntga tntataaatg cggnggcatcgacanaanaa 120 ccatngnaan atttganaag gaggctgctg atatnggaaa gggctccntcnantntgcct 180 gggtcttgga tnaactgaaa nctgancntg aacgtggnnt caccattgatatctncttgt 240 ggaaatntna gaccancann tactatgtna ctatcattga tgccccaggacacaganact 300 ttatcnaaan catgattacn nggacatnta nagctgactg tgctngcctgattgtngctg 360 ctggtgttgg tgaatttgaa nctggtatnt ccaana 396 50 396 DNAHomo sapien 50 cgacttcttg ctggtgggtg gggcagtttg gtttagtgtt atactttggtctaagtattt 60 gagttaaact gcttttttgc taatgagtgg gctggttgtt agcaggtttgtttttcctgc 120 tgttgattgt tactagtggc attaactttt agaatttggg ctggtgagattaattttttt 180 taatatccca gctagagata tggcctttaa ctgacctaaa gaggtgtgttgtgatttaat 240 tttttcccgt tcctttttct tcagtaaacc caacaatagt ctaaccttaaaaattgagtt 300 gatgtcctta taggtcacta cccctaaata aacctgaagc aggtgttttctcttggacat 360 actaaaaaat acctaaaagg aagcttagat gggctg 396 51 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 51 ttttttttttttcagcgngg atttatttta tttcattttt tactctcaag anaaagaana 60 gttactattgcaggaacaga cattttttta aaaagcgaaa ctcctgacac ccttaaaaca 120 gaaaacattgttattcacat aataatgngg ggctctgtct ctgccgacag gggctgggtt 180 cgggcattagctgtgccgtc gacaatagcc ccattcaccc cattcataaa tgctgctgct 240 acaggaagggaacagcggct ctcccanaga gggatccacc ctggaacacg agtcacctcc 300 aaagagctgcgactgtttga naatctgcca anaggaaaac cactcaatgg gacctggata 360 acccaggcccgggagtcata gcaggatgtg gtactt 396 52 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 52 acctcgctaa gtgttcgcta cgcggggctaccggatcggt cggaaatggc agaggtggag 60 gagacactga agcgactgca nagccagaagggagtgcagg gaatcatcgt cgtgaacaca 120 gaaggcattc ccatcaagag caccatggacaaccccacca ccacccagta tgccagcctc 180 atgcacagnt tcatcctgaa ggcacggagcaccgtgcgtg acatcgaccc ccagaacgat 240 ctcaccttcc ttcgaattcg ctccaagaaaaatgaaatta tggttgcacc agataaagac 300 tatttcctga ttgtgattca gaatccaaccgaataagcca ctctcttggc tccctgtgtc 360 attccttaat ttaatgcccc ccaagaatgttaatgt 396 53 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 53 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180tttttttttt tttttttttt tttttttttt tttttttttt ttannttntt ttttnttttn 240cctttntttt aattcanaaa aagaanaaga aaanataana nnnancnnan nnnnnnnatn 300ntncttnata ntnnttnnnn nanngggnnn gcgagnnnnn nnnnnnnnnn nntctnnnnt 360tnnnnnnctt gcnccccttn nnttngnnnn angcaa 396 54 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 54 ctcttggggc tgctgggactcgcgtcggtt ggcgactccc ggacgtaggt agtttgttgg 60 gccgggttct gaggccttgcttctctttac ttttccactc taggccacga tgccgcagta 120 ccagacctgg gaggagttcagccgcgctgc cgagaagctt tacctcgctg accctatgaa 180 ggcacgtgtg gttctcaaatataggcattc tgatgggaac ttgtgtgtta aagtaacaga 240 tgatttagtt tgtttggtgtataaaacaga ccaagctcaa gatgtaaaga agattgagaa 300 attccacagt caactaatgcgacttatggt agccaaggaa gcccgcaatg ttaccatgga 360 aactgantga atggtttgaaatgaagactt tgtcgt 396 55 396 DNA Homo sapien 55 cgacggtttg ccgccagaacacaggtgtcg tgaaaactac ccctaaaagc caaaatggga 60 aaggaaaaga ctcatatcaacattgtcgtc attggacacg tagattcggg caagtccacc 120 actactggcc atctgatctataaatgcggt ggcatcgaca aaagaaccat tgaaaaattt 180 gagaaggagg ctgctgagatgggaaagggc tccttcaagt atgcctgggt cttggataaa 240 ctgaaagctg agcgtgaacgtggtatcacc attgatatct ccttgtggaa atttgagacc 300 agcaagtact atgtgactatcattgatgcc ccaggacaca gagactttat caaaaacatg 360 attacaggga catctcaggctgactgtgct gtcctg 396 56 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 56 tttttttttt ttttttctca tttaactttt ttaatgggtc tcaaaattctgtgacaaatt 60 tttggtcaag ttgtttccat taaaaagtac tgattttaaa aactaataacttaaaactgc 120 cacacgcaaa aaanaaaacc aaagnggtcc acaaaacatt ctcctttccttctgaaggtt 180 ttacgatgca ttgttatcat taaccagtct tttactacta aacttaaatggccaattgaa 240 acaaacagtt ctganaccgt tcttccacca ctgattaana gtggggtggcaggtattagg 300 gataatattc atttagcctt ctgagctttc tgggcanact tggngaccttgccagctcca 360 gcagccttnt tgtccactgc tttgatgaca cccacc 396 57 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 57 cctttttttttttttttttt tttttttttt tttttttttt tttttttttt tnaaaanntt 60 ntttttgcaaanccnancaa aaanggnngg aangaaaaan nggaaaaatt ntttttncnt 120 ntttgggaacnnnnagccct tnntttgaaa aaangnggnc ttaaaanngn tgaannaaag 180 gnnanncccngntncttnnn tttaaaaana anggggnngn ttttttttaa anaanatttt 240 ttttttccctaanancnncn anntgaaacn ngncccnacn nctnncttna aagggnnnaa 300 atnanangnnaaaaaanccc tnancccccc cccttanntt tncnannana naaagncntt 360 ttgggncntgnaaaaanaan cctttttnnt gcnttn 396 58 396 DNA Homo sapien 58 cgacctcaaatatgccttat tttgcacaaa agactgccaa ggacatgacc agcagctggc 60 tacagcctcgatttatattt ctgtttgtgg tgaactgatt ttttttaaac caaagtttag 120 aaagaggtttttgaaatgcc tatggtttct ttgaatggta aacttgagca tcttttcact 180 ttccagtagtcagcaaagag cagtttgaat tttcttgtcg cttcctatca aaatattcag 240 agactcgagcacagcaccca gacttcatgc gcccgtggaa tgctcaccac atgttggtcg 300 aagcggccgaccactgactt tgtgacttag gcggctgtgt tgcctatgta gagaacacgc 360 ttcacccccactccccgtac agtgcgcaca ggcttt 396 59 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 59 cttttttttt tttttttttt tcagnggaaaataactttta ttganacccc accaactgca 60 aaatctgttc ctggcattaa gctccttcttcctttgcaat tcggtctttc ttcagnggtc 120 ccatgaatgc tttcttctcc tccatggtctggaagcggcc atggccaaac ttggaggngg 180 tgtcaatgaa cttaaggnca atcttctccanagcccgccg cttcntctgc accancaagg 240 acttgcggag ggngagcacc cgcttnttggttcccaccac ncagcctttc agcatgacaa 300 agtcattggt cacttcacca tagnggacaaagccacccaa agggttgatg ctccttggca 360 aataggncat agtcacngga ggcattgtncttgatc 396 60 396 DNA Homo sapien 60 acctcagctc tcggcgcacg gcccagcttccttcaaaatg tctactgttc acgaaatcct 60 gtgcaagctc agcttggagg gtgatcactctacaccccca agtgcatatg ggtctgtcaa 120 agcctatact aactttgatg ctgagcgggatgctttgaac attgaaacag ccatcaagac 180 caaaggtgtg gatgaggtca ccattgtcaacattttgacc aaccgcagca atgcacagag 240 acaggatatt gccttcgcct accagagaaggaccaaaaag gaacttgcat cagcactgaa 300 gtcagcctta tctggccacc tggagacggtgattttgggc ctattgaaga cacctgctca 360 gtatgacgct tctgagctaa aagcttccatgaaggg 396 61 396 DNA Homo sapien 61 tagcttgtcg gggacggtaa ccgggacccggtgtctgctc ctgtcgcctt cgcctcctaa 60 tccctagcca ctatgcgtga gtgcatctccatccacgttg gccaggctgg tgtccagatt 120 ggcaatgcct gctgggagct ctactgcctggaacacggca tccagcccga tggccagatg 180 ccaagtgaca agaccattgg gggaggagatgactccttca acaccttctt cagtgagacg 240 ggcgctggca agcacgtgcc ccgggctgtgtttgtagact tggaacccac agtcattgat 300 gaagttcgca ctggcaccta ccgccagctcttccaccctg agcagctcat cacaggcaag 360 gaagatgctg ccaataacta tgcccgagggcactac 396 62 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 62 tcgacgtttc ctaaagaaaa ccactctttg atcatggctc tctctgccag aattgtgtgc60 actctgtaac atctttgtgg tagtcctgtt ttcctaataa ctttgttact gtgctgtgaa 120agattacaga tttgaacatg tagtgtacgt gctgttgagt tgtgaactgg tgggccgtat 180gtaacagctg accaacgtga agatactggt acttgatagc ctcttaagga aaatttgctt 240ccaaatttta agctggaaag ncactggant aactttaaaa aagaattaca atacatggct 300ttttagaatt tcnttacgta tgttaagatt tgngtacaaa ttgaantgtc tgtnctganc 360ctcaaccaat aaaatctcag tttatgaaan aaannn 396 63 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 63 ttnttttttt nttttntnttttntcnttgn ttgnacngaa cccggcgctn nttccccacn 60 nnnnacggcc gcccntattcannnntncnt canntannna ccgcaccctc ggactgcnnn 120 tngggccccg ccgncnanncnccnncnccc anttcnccgc cgccgccgcc gccttttttt 180 attggcnncc atnanaaccggggncacctc ncangngcgc cnaaantngg ggcangactc 240 anagggggcc atcaaccnccaagnncaanc tgganctcta caaacggcct acgntttntg 300 nccatgnggg tagggntttacccgcnatga tgannatgnn aanaactttn ncaanccctt 360 tattaaccaa tgnggtgnggagacggaacn tggtta 396 64 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 64 tcgacgtcgg ggtttcctgc ttcaacagtg cttggacgga acccggcgctcgttccccac 60 cccggccggc cgcccatagc cagccctccg tcacctcttc accgcaccctcggactgccc 120 caaggccccc gccgccgctc cagcgccgcg cagccaccgc cgccgccgccgcctntnctt 180 agtcgccgcc atgacgaccg cgtccacctc gcaggtgcgc cagaactaccaccaggactc 240 agaggccgcc atcaaccgcc agatcaacct ggagctctac gcctcctacgtttacctgtc 300 catgtcttac tactttgacc gcgatgatgt ggctttgaan aactttgccaaatactttct 360 tcccaatctc atgaggagaa ggaacatgct ganaaa 396 65 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 65 tttttttttttttttttttt tttttnacca ataatgcttt tattttccac atcaanatta 60 atttatatgttagttttagt acaagtacta aaatgtatac ttnttgccct aatagctaag 120 gnatacataagcttcaccat acatnttgca nccncctgtc tgtcctatgt cattgttata 180 aatgtananattttaggaaa ctnttttatt caacctggga catntatact gtaggagtta 240 gcactgacctgatgtnttat ttaaaagtaa tgnatattac ctttacatat attccttata 300 tattnaaacgtatttccatg ttatccagct taaaatcaca tggnggttaa aagcatgagt 360 tctgagtcaaatctggactg aaatcctgat gctccc 396 66 396 DNA Homo sapien 66 tcgacttttttttttccagg acattgtcat aattttttat tatgtatcaa attgtcttca 60 atataagttacaacttgatt aaagttgata gacatttgta tctatttaaa gacaaaaaaa 120 ttcttttatgtacaatatct tgtctagagt ctagcaaata tagtaccttt cattgcagga 180 tttctgcttaatataacaag caaaaacaaa caactgaaaa aatataaacc aaagcaaacc 240 aaaccccccgctcaactaca aatgtcaata ttgaatgaag cattaaaaga caaacataaa 300 gtaacttcagcttttatcta gcaatgcaga atgaatacta aaattagtgg caaaaaaaca 360 aacaacaaacaacaaacaaa acaaaacaaa caaaca 396 67 396 DNA Homo sapien 67 acgcttttgtccttcatttt aactgttatg tcatactgtt atgttgacat atttctttat 60 aagagaatagaggcaaaagt atagaactga ggatcatttg tatttttgag ttggaaatta 120 tgaaacttcaccatattatg atcatacata ttttgaagaa cagactgacc aaagctcacc 180 tgttttttgtgttaggtgct ttggctgaac ttgattccag cccccttttc cctttggtgt 240 tgtgtatgtctcttcatttc ctctcaaatc ttcaactctt gccccatgtc tccttggcag 300 caggatgctggcatctgtgt agtcctcata ctgtttactg ataacccaca aattcatttt 360 catggcagacctaagctcag accctgcctt gtcctg 396 68 396 DNA Homo sapien 68 acctgagtcctgtcctttct ctctccccgg acagcatgag cttcaccact cgctccacct 60 tctccaccaactaccggtcc ctgggctctg tccaggcgcc cagctacggc gcccggccgg 120 tcagcagcgcggccagcgtc tatgcaggcg ctgggggctc tggttcccgg atctccgtgt 180 cccgctccaccagcttcagg ggcggcatgg ggtccggggg cctggccacc gggatagccg 240 ggggtctggcaggaatggga ggcatccaga acgagaagga gaccatgcaa agcctgaacg 300 accgcctggcctcttacctg gacagagtga ggagcctgga gaccgagaac cggaggctgg 360 agagcaaaatccgggagcac ttggagaaga agggac 396 69 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 69 ntcncngnng ntgtggtnnt ttttttaatttttatntttt cttttttttt ctngctagcn 60 cttncttttt ttggaattnc ggtncctttttntntcnatt ttttngacaa aaanaacctn 120 ttntttnana ccanagnnng gnncacncntnnaatntncc ccttttncgn tngggagctn 180 cncnttnnnc gccnacntca ntcgagacngtncttttnnn tnnancannn tnngtncgtt 240 gncngcnttn ntncannant nttccctatnnacntgnnnt cncncatnnt tggacnancn 300 cctagccttn ccatnntttn nttntttntnnatnancctn gaaaacntcn gnntnttcnc 360 nncnttnccn cncncncctt cntatgtncnatgncn 396 70 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 70 tttttttttt ttttnttttt tttttttttt tttttttntt tttttttttt ttttttntnc60 aannnntnaa cttttaanng gccnccngcn ccccaanggg gaccctgctt ttgnnggcta 120aatgccnnaa aactttgggg nantnggtat naaaccccnc tttgcccnnc annttncngg 180gggggggggg tttttgnngg ggaacangna naacnttttn ncnanggnat caccaaaaan 240aaagcccnnc cctttttccn annggggggg ggngggggga aantcanccc ccanattgac 300cttnatttca aaanggggct tataatcctg ggcntggann cttccctnta cccgggggtt 360gnccacnttt tattanaggg gnangnggat ccccnt 396 71 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 71 gcatctagag ggccngtttantctagaggn ccngnntaaa cnnnnncatc nacctncnnt 60 gcncctgctn gttgccncccntctgtgnct tgcnnnnccc nngagcgtnc cttnaccnnn 120 gaangtgcct nnnnnactgannnnnncnna taanatgngg anantncgtc gncattntnt 180 natnnggggt gatgctattctggggggtgg ggnggngnna tnnnatactn nggggacgtn 240 nnatnangag nnatntcnngnttntctnnt gntttntggg gggcnatnng nnntctntnn 300 ggactcntcg cncannnatcaatancttna ttcngtgtan ngtccgnccn tagnncngcn 360 ngtactnnan ngttgnnntcattactnttc gtnngg 396 72 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 72 tntttttttt tttctaaaac atnactnttt attnnnnang ntttntgaacctctnngcnt 60 natggtgaga gtttgtctga ttaataanaa tnggannntt nannanangcntgnncgcaa 120 ngatggcnnc nctgtatatc ccaccatccc attacactnt gaaccttttntttgattaat 180 aaaaggaagg natgcgggga anggggaaag agaatgcttg aacattnccatgngnccttn 240 gacaaacttt ccaatggagg cnggaacnaa nnaccaccan ncaactcccctttttgtaat 300 ttnnnaactt ncaacnncta nctntttatt ttggcntccc tggnngaaacagnctgtatn 360 annnnnaagn ccntgagaac atccctggnt nncnna 396 73 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 73 ntcaacntngactnctgtga ggnatggtgc tgggngcnta tgcngtgngn ttttggatac 60 naccttatggacantngcnn tcccnnggaa ngatnataat ncttactgna gnnactnnaa 120 nnttccntntcnaaaangtt naaaancatt ggatgtgcca caatgatgac agtttatttg 180 ctactcttgagtgctataat gatgaagatc ttanccacca ttatcttaac tgangcaccc 240 aanatggtganttggggaac atatanagta cacctaagtt cacatgaagt tgtttnttcc 300 caggnnctaaagagcaagcc taactcaagc cattgncaca caggtgagac acctctattt 360 tgtacttctcacttttaagg gattagaaaa tagcca 396 74 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 74 cctttttttt tttttttact gngaatatatactttttatt tagtcatttt tgtttacaat 60 tgaaactctg ggaattcaaa attaacatccttgcccgtga gcttcttata gacaccanaa 120 aaagtttcaa ccttgtgttc cacattgttctgctgtgctt tgtccaaatg aacctttatg 180 agccggctgc catctagttt gacgcggattctcttgccca caatttcgct tgggaagacc 240 aagtcctcaa ggatggcatc gtgcacagctgtcagagtac ggctcctggg acgcttttgc 300 ttattttttg tacggctttt tcgagttggcttaggcagaa ttctcctctg agcgataaag 360 acgacatgct tcccactgaa ctttttctccaattcg 396 75 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 75 tttttttttt tttntttttt tttttttttt tttttttnaa ntntaanggg ganggcccct60 tttttttaaa ctngnccntt ttnctttcct tttttnaaaa ggaaaaaaaa anntttnttt 120ttcnttnaaa aacccttttt cccacnaaca aaaaaaaccn ttccccntnc cttttnnnna 180aaaaaaaggg gctnggnntt tccccttann caaaaaaccn tntccnnggg naaaaaantt 240ntcnccgggg gggaaacnnn tgggggtgtn nccnaaattt gggggccntc ggaagggggg 300nnccncncct aaagangtnt ttcaaaanaa aaacccccnt cctnttntaa aaanaaaana 360aaanaangnn ngnntttttt ntcnttnncc ccccaa 396 76 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 76 acattcttca gaaatacagtgatgaaaatt cattttgaaa ctcaaatatt ttcattttgg 60 atattctcct gtttttattaaaccagngat tacncctggc cntccctnta aatgttctag 120 gaaggcatgt ctgttgtnntttnnnnaaaa nnaaattntt tttttttngn naaaccccaa 180 atcccanttt atcaggaagttagncnaatg aaatggaaat tggntaatgg acaaaagcta 240 gcttgtaaaa aggaccacccnnccacnngn ctttaccccc ttggttngtt gggggaaaaa 300 ccatnnttaa ccntntggnnaaaattgggn ncntaaagtt tncntggnna acagtncntn 360 cngtattnaa ttgncnttatnggaaaatcn gggatt 396 77 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 77 tttttttttt tttttttttt tttttttttt tatcaacatt tatatgctttattgaaagtt 60 ganaanggca acagttaaat ncngggacnc cttacaattg tgtaaanaacatgcncanaa 120 acatatgcat ataactacta tacaggngat ntgcaaaaac ccctactgggaaatccattt 180 cattagttan aactgagcat ttttcaaagt attcaaccag ctcaattgaaanacttcagt 240 gaacaaggat ttacttcagc gtattcagca gctanatttc aaattacncaaagngagtaa 300 ctgngccaaa ttcttaaaat ttntttaggg gnggtttttg gcatgtaccagtttttatgt 360 aaatctatnt ataaaagtcc acacctcctc anacag 396 78 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 78 agctggcnaaaggngnatgn gctgcnangc gattangnnn ggtaacgtca nnggntnncc 60 agtgcangacnttgtaaaac gacggccaca tgaattgtaa tacgactcac tatngggcgn 120 attgggccgtgnaggatngt gntcacactc gaatgtatnc tggcngatnc ananngcttt 180 atngctnttgacggngnntn anccanctng ggctttaggg ggtatcccct cgcccctgct 240 tcnttgatttgcacgggcnn ctccganttc cttcataata ccngacgctt cnatccccta 300 gctcngacctntcantntnt tcnntgggtt ntnnccgntc acngcttncc cgnangntat 360 aatctnggctcctttnggga tccattantc tttact 396 79 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 79 caccaaccaa aacctggcgc cgttggcatcgtagagtgaa cacaacccaa aaacgatacg 60 ccatctgttc tgccctggct gcctcagccctaccagcact ggtcatgtct aaaggncatc 120 gtattgagga agttcctgaa cttcctttggtangttgaag ataaagctga aggctacaag 180 aagaccaang aagntgtttt gctccttaanaaacttanac gcctggaatg atatcaaaaa 240 ngctatgcct ctcagcgaat gagactgganangcaaaatg agaaaccntc nccgcatcca 300 gcgnaggggc cgtgcatctc tatnntgangatnntggnan cnttcaaggc cttcagaacc 360 tccctngaaa tnctctnctt taangaaccaaactgn 396 80 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 80 tgtacatagg catcttattc actgcaccct gtcacaccca gcaccccccg ccccgcacat60 tatttgaaag actgggaatt taatggttag ggacagtaaa tctacttctt tttccaggga 120cgactgtccc ctctaaagtt aaagtcaata caagaaaact gtctattttt agcctaaagt 180aaaggctgtg aagaaaattc attttacatt gggtagacag taaaaaacaa gtaaaataac 240ttgacatgag cacctttaga tccttccctt catggggctt tgggcccaga atgacctttg 300aggcctgtaa anggattgna atttcctata agctgtatag tggagggatt ggngggtcat 360ttgagtaagc cctccaagat acnttcaata cctggg 396 81 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 81 gcagctgaag ttcagcaggtgctgaatcga ttctcctcgg cccctctcat tccacttcca 60 acccctccca ttattccagtactacctcag caatttgtgc cccctacaaa tgttagagac 120 tgtatacgcc ttcgaggtcttccctatgca gccacaattg aggacatcct gcatttcctg 180 ggggagttcg ccacagatattcgtactcat ggggttcaca tggttttgaa tcaccagggn 240 ccgccatcag gagatgcctttatccagatg aagtctgcgg acagancatt tatggctgca 300 cagaagtggc ataaaaaaaacatgaaggac agatatgttg aagttttcag tgtcagctga 360 nganagaaca ttgnngtannngggggnact ttaaat 396 82 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 82 gactcagaaa tgtcagtctc atgaagttca aaagatcgag aatgtttgctatcttggtgg 60 agcagccgca gccaagcaag taacttgtaa aatgaggaat gccatcacccctcgagtgtc 120 catcccacat aacttggggt tagagcacaa gcgttcccag gaactactcaccttaccatc 180 ttggccgttt catttgcttc caccagttct ggaaagagan ggcctagaagttcaaaaaaa 240 aagtaggaaa ngtgcttttg gagaaaatca cctgctcctc agaactgggcttacaanctg 300 ngaagtacnc tatgtgccac ctaatcctca tatatgacct caagagacnccaataagcat 360 atttccacca cggaatgacc agtgctttgg gtaana 396 83 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 83 tttgatttaaganatttatt atttttttaa aaaaagcaac ttccagggtt gtcattgtac 60 aggttttgcccagtctccta tagcatggta tagtgataac tgatttttta taacaatgac 120 tcagaggcattgaagatcca taactatctt ctgaattatc acagaaagaa gaaagttaga 180 agagtttaatgttaagtgta ttaaaaatca tattctaatt cttttaattt ggttatctga 240 gtatgataatataggagagc tcagataaca aggaaaaggc attggggtaa gaacactcct 300 tcccacaggatggcattaac agactttttc tgcatatgct ttatatagtt gccaactaat 360 tcaccttttacncagcttna ttttttttta ctnggg 396 84 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 84 tttttacagc aatttttttt tattgatgtttaacctgtat acaaccatac ccattttaag 60 ngtacagaca aatgaatttt gacaaattcattcactcatc taatcatcac tataaccatg 120 atacagattt ttatcactcc aaaagtccatcctgtgctct tttcaagtcc atcctcctca 180 tctgataccc caagccacca ttgttttgctttctggaact acagttttgg gnttttagaa 240 tttcatatat ggtngaatca taccatttgnnatttggggc tgacgncttt cctccaataa 300 tggatttgag aattatctac attttgcatggatcctgggt tatttatacc aacnangggt 360 tattatgnaa aatnggacca caatttggnggcanta 396 85 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 85 cagtgaccgt gctcctaccc agctctgctc cacagcgccc acctgtctcc gcccctcggc60 ccctcgcccg gctttgccta accgccacga tgatgttctc gggcttcaac gcagactacg 120aggcgtcatc ctcccgctgc agcagcgcgt ccccggccgg ggatagcctc tcttactacc 180actcacccgc agactccttc tccagcatgg gctcgcctgc aacgcgcagg acttctgcac 240ggacctggcc gctccagtgc caacttcatt ccacggcact gcatctcgac canccggact 300tgcannggtt ggggaanccg cccttgtttc tccgtggccc atctaanacc aaacccntca 360ccttttcgga gnccccnccc ctccgntggg nttact 396 86 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 86 ttttnnactg aatgtttaatacatttgnag gaacagaaga aatgcagtan ggattaanat 60 tttataatta gacattaatgtaacagatgn ttcatttttc aaagaagntn cccccttntc 120 cctatctttt tttaatcttccttanagcaa taantagtaa ttactatatt tgtggacaag 180 ctgctccact gtgntggacagtaattatta aatctttatg tttcacatca ttattacctt 240 ccanaattct accttcatttccctgcacag gttcactgga ctggntcaca ancaaattgn 300 actccactca antanaagagcccaaagaaa ttagagtaac gncnantcct atgaattana 360 gacccaaaga tttnaggngntgattagaaa cataan 396 87 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 87 atggaggcgc tggggaagct gaagcagttc gatgcctacc ccaagactttggaggacttc 60 cgggtcaaga cctgcggggg cgccaccgtg accattgtca gtggccttctcatgctgcta 120 ctgttcctgt ccgagctgca gtattacctc accacggagg tgcatcctgagctctacgtg 180 gacaagtcgc ggggagataa actgaagatc aacatcgatg tactttttccncacatgcct 240 tgtgcctatc tgagtattga tgccatggat gtggccngag aacancagctggatgnggaa 300 cacaacctgt ttaagccacc actagataaa gatgcatccc ngtgagctcanagctgagcg 360 gcatgagctt gngaaantcn aggtgaccgg gtttga 396 88 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 88 tccagagcagagtcagccag catgaccgag cgccgcgtcc ccttctcgct cctgcggggc 60 cccagctgggaccccttccg cgactggtac ccgcatagcc gctcttcgac caggccttcg 120 ggctgccccggctgccggag gagtggtcgc agtggttagg cggcagcagc tggccaggct 180 acgtgcgccccctgcccccc gccgcatcga gagccccgca gtggccgcgc ccgctacagc 240 cgcgcngctcagccggcaac tcacancggg gctcggagat ccgggacact gcggaccgct 300 ngcgcgtgccctggatgtca ccactttngc ccggacaact gacggtnana caaggatggg 360 gggtgganannccngtaanc caagaanggg naggac 396 89 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 89 gagagaacag taaacatcca gccttagcatctctcangag tactgcagat cttcattagc 60 tatattcaca tggagnaatg ctattcaacctatttctctt atcaaaacta attttgtatt 120 ctttgaccaa tgttcctaaa ttcactctgcttctctatct caatcttttt cccctttctc 180 atctttcctc cttttttcag tttctaactttcactggttc tttggaatgn tttttctttc 240 atctcttttc ttttacattt tggggtgtcccctctctttt cttaccctct ttctncatcc 300 ttcttnttct tttgaattgg ctgccctttatcntctcatc tgctgncatc ttcatttctc 360 ctccctcctn tttccnntca ttctactctctcccnt 396 90 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 90 gggcgccggc gcgccccccc acccccgccc cacgtctcgt cgcgcgcgcg tccgctgggg60 gcggggagcg gtcgggccgg cngcggtcgg ccggcggcag ggtggtgcgn tttcnttttn 120nattnnccnc nttcttcttn nttnnncnnn ctnntanncn ntnncnttcn cnnnntttnc 180tntntcttna ccnnnttttn taatcntctt ctncntnnnn tctcttnnat ntnttnctta 240nttcctnnnn tttnttctnt cntttctcnc ctnnntctcn nnctcnncnc tcnncatttt 300nntnttttnt nccttctnnt cttnnttctn ntnntnnttt nnnnttctnt tnntcatntt 360ncctntntta ctntcanctt ntatnnncct cntttt 396 91 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 91 ntntcctnna tttttnnntcnncttttttt tnnaattttt ctttnttttn tttataaaaa 60 tcnncacnta aaacngcggaanaggggatt tnttnttngg gngtancncn nggccncaaa 120 naaccccaaa aatancccaaaatgcacagg nccngggnaa angaccnacn tgggtntttt 180 ntttntnaac aaggggggttttaaagggna tnggnatcaa agggnataaa ntttaaacct 240 ttganaaatt ttttaanaggcttgcccccc actttggncc ccnccccncn gnngggatcc 300 aatttttttt cnttggggctcccngncccn nannttccgg gttnntggnc nntcctnntt 360 tttttttttt tgccttcacccntnccattn cntttt 396 92 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 92 ctntttnnnt ntttttttcc ccatcatcca naaatgggtt ttattctcagccgagggaca 60 gcaggactgg taaaaactgt caggccacac ggttgcctgc acagcacccccatgcttggt 120 agggggtggg agggatggcg ggggctggnt gnccacaggc cgggcatgacaaggaggctc 180 actggaggtg gcacactttg gagtgggatg tcgggggaca ncttctttggtanttgggcc 240 acaagattcc caaggatanc acnnnnactg attnccannc tanagncaagcggntggcca 300 tntgtangnn nttntntatn tgactattta tagattttta tanaacagggnaagggcata 360 ccncaaaagg gnccaanttt ttaccnccgg gcnccc 396 93 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 93 gctgccacagatctgttcct ttgtccgttt ttgggatcca caggccctat gtatttgaag 60 ggaaatgtgtatggctcaga tcctttttga aacatatcat acaggttgca gtcctgaccc 120 aagaacagttttaatggacc actatgagcc cagttacata aagaaaaagg agtgctaccc 180 atgttctcatccttcagaag aatcctgcga acggagcttc agtaatatat cgtggcttca 240 catgtgaggaagctacttaa cactagttac tctcacaatg aaggacctgn aatgaaaaat 300 ctgnttctaaccnagtcctn tttanatttt agngcanatc cagaccancg ncggtgctcg 360 agtaattctttcatgggacc tttggaaaac tttcag 396 94 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 94 tgccttaacc agtctctcaa gtgatgagacagtgaagtaa aattgagtgc actaaacgaa 60 taagattctg aggaagtctt atcttctgcagtgagtatgg cccaatgctt tctgnggcta 120 aacagatgta atgggaagaa ataaaagcctacgtgttggt aaatccaaca gcaagggaga 180 tttttgaatc ataataactc atanngtgctatctgtcagt gatgccctca gagctcttgc 240 tgntagctgg cagctgacgc ttctangatagttagnttgg aaatggtctt cataataact 300 acacaaggaa agtcanccnc cgggcttatgaggaattgga cttaataaat ttagngngct 360 tccnacctaa aatatatctt ttggaagtaaaattta 396 95 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 95 cctcccaccc ncttanttca tgagattcga naatgncact tntgtgctnt ttnctnnttn60 tattctnacn atttctttct tggngcggna nnaatcccnt ttttnngggc gnctctcccn 120ncttntnntt tcntggngct ntcccttttc nnnnnaaact tntacnnngt ttanaantnt 180ttctgnangg gggnntccna aananttttt ccncctncct nattccnctc tnaannctcn 240cnaattgttt cccccccccn ntagnntatt ttttctaaaa aattaactcc nacgganaaa 300attttcccta aaatttcncc tccanatttn gaaaaaacnc gcccgganct nntntncgaa 360tntnaatttt tnaaaaaaan ttattttcat cnggnn 396 96 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 96 cctgggtacc aaatttctttatttgaagga atggtacaaa tcaaagaact taagtggatg 60 ttttggacaa cttatagaaaaggtaaagga aaccccaaca tgcatgcact gccttggcga 120 ccagggaagt caccccacggctatggggaa attagcccga ngcttaactt tcattatcac 180 tgcttccaag ggngtgcttggcaaaaaaat attccgccaa ccaaatcggg cgctccatct 240 tgcccagttg gtnccgggnccccaattctt ggatgctttc ncctcttntt ccggaatgng 300 ctcatgaant cccccaannggggcattttg ccagnggccn tttngccatt cnagnnggcc 360 tgatccattt tttccaatgtaatgccnctt cattgn 396 97 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 97 ctcaccctcc tcntnnttnt canaatattg ngaacttnnt nctgntcgaatcactggcat 60 taaagganca ctagctaatg gcactaaatt tacnnactan ggaaacttttttataatant 120 gcaaaaacat ntnaaaaaga ntgnagttcg cccatttctg cttnggaaganctcttcact 180 tntaancccn natgnngncc tttgggtcaa aanctccgcg attattacngngttncccnc 240 tatttgncct tcctttntcc ccaangccnc anatttcnna actttnccntnaaatgcctt 300 tatttnatnn cntttcnacn ncttaanntt ccctttnaan aangatccctncttcaaatn 360 ntttcccngt tcctngcatt ncccnnnnat ttctct 396 98 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 98 acagggacaatgaagccttt gaagtgccag tctatgaaga ggccgtggtg ggactagaat 60 cccagtgccgcccccaagag ttggaccaac caccccctac agcactgttg tgataccccc 120 agcacctgangaggaacaac ctaccatcca gaggggccag gaaaagccaa actggaacag 180 aggcgaatggctcagagggg tncatggcca agaaggaagc cctggaagaa cttcaatcac 240 cttcggtttcgggaccaccg gcttgtgtcc ctgttctgac tgcanaactt ggcgcngtnc 300 cccattanaacctntgactc nncccttgct ataagnctgt tttggcccct gatgatgata 360 gggtttttatgangacactt gggcaccccc ttaatg 396 99 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 99 nttntttttc cgncnaaagg gcaagngtttncatctttcc tgnccncnca ananngggtn 60 tntgtgcntt tnttttttcc caaaacccgggtnggggaca ccttttgagg anccactnnt 120 cntccggggc nnnnttttag aaggngnctaanaagcntct tgnnggggga aaaacatctt 180 tttgcncccn acataccccc aaggggggggggtgtctggg agganactaa ngacttttnt 240 tttttnnccn caaanaactg anggcccccattgctccccc cccantcttt aaaaaacccc 300 ttcaatttcc ttgncnggna aaaanggttggnaaaaaang agngngcntc nnttncnttt 360 natggaaggn aaaaggtttt tggttgnaaaaccccg 396 100 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 100 ctaacacggt gaaaccctgt ctctactaaa aatacaaaaa aattagccag gcgtggtggc60 gggcacctgt agtcccagct gctcaggaag ctgaggcagg agaatggcgt gaacccagaa 120ggcggagctt gcagtgagct gagatcgtgt cagtgcactc cagcctgggc gacagagcga 180gactcccgct caaaaaaaaa aaaaaaaaga gaaaagaaaa agctgcagng agctgggaat 240gggccctatc ccctccttgg ggatcaatga gacccctttt caaaanaaaa aaaaaaataa 300tgngattttg gnaacatatg gcactggtgc ttcnnggaat tctgtttntn ggcatgnccc 360cctntgactg nggaaaaatc cagcaggagg cccana 396 101 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 101 agttataact caacagttcatttatatgct gttcatttaa cagttcattt aaacagttca 60 ttataactgt ttaaaaatatatatgcttat agncaaaann tgttgtggcg nagttgttgc 120 cgcttatagc tgagcattatttcttaaatt cttgaatgtt cttttggngg gntnctaaaa 180 ccgtatatga tccattttnatgggaaacng aattcntnnc attatcncac cttggaaata 240 cnnaacgtgg gggaaaaaaatcattcccnc cntccaaaac tatacttctt ttatctngan 300 nttcttgntc ctgcncnggtttngaatata nctgggcaaa nggntttncc aaatccntnt 360 acnntncttt gggaantancggcaantcnt cncttt 396 102 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 102 actatacata agaacangct cacatgggag gctggaggtg ggtacccagctgctgtggaa 60 cgggtatgga caggtcataa acctagagtc agngtcctgt tggcctagcccatttcagca 120 ccctgccact tggagnggac ccctctactc ttcttagcgc ctaccctcatacctatctcc 180 ctnctcccat ctcctacgga ctggcgccaa atggctttcc tgccaattttgggatcttct 240 ctggctctcc agcctgctta ctcctctatt tttaaagggc caaacaaatcccttctcttt 300 ctcaaacaca gtaatgnggc actgacccta ccacacctca tgaagggggcttgttgcttt 360 tatttgggcc cgatctgggg ggggcaaaat attttg 396 103 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 103 ttgtgttgggactgctgata ggaagatgtc ttcaggaaat gctaaaattg ggcaccctgc 60 cccaacttcaaagccacagc tggtatgcca natggtcagg ttaaagatat caacctgctg 120 actacaaaggaaaatatggt ggggtcttct tttaccctct tgacttccct ttgngngccc 180 cccgagancattgctttccg ngatagggca aaanaaatta aaaaacttaa ctggccagtg 240 aatggggcttctgnggatct ccttctggca ttacatnggc aatccctaaa aaacaagang 300 actgggacccataacattct tttgnatcaa ccgaagcccc cattgttang atatngggct 360 taaangctgatnaagcatct cgtccgggcn ttttat 396 104 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 104 aagggagggc gcgccaagac cttcccactcgngcacactg ggggcgccga cangacgcaa 60 cccagtccaa cttggatacc cttggntttagttctcggac acttctttta tctctccgtc 120 gcaacttgtc aagttctcaa nactgtctctctgngntatc ttttttcttc gctgctcttc 180 nncccccgac gtatttntca aaangtctgcaattgttgna tacntnganc tncaccactg 240 ttacnaggtc atnaatttcn cntcaactctntnccncttg ttccctgata tntcggccgg 300 ngncnccaat tctgtatttt nctcntcaacgntctcactt ttncctcctc cnggccactt 360 tctccccttc cttattccgg cnttgtttgccnccat 396 105 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 105 tcaatagcca gccagtgttc atttttatcc ttgagctttt agtaaaaact tcctggnttt60 atttttagtc attgggtcat acagcactaa agtctgctat ttatggaaac taactttttt 120gtttttaatc caggccaaca tgtatgtaaa ttaaattttt agataattga ttatctcttt 180gtactacttg agatttgatt atgagatgtg catattgctt tgggaagagc tcgaggaagg 240aaataattct ctcctttggt ttgaacctca actagataaa ccctaggaat tgttaactgc 300acaagnattt tcattccaca aaacctgagg cagctctttt gccagagcgt tcctgnaccc 360ccccacccca cttgccttgg gtctttanaa ngagcc 396 106 396 DNA Homo sapien 106gctgtgtagc acactgagtg acgcaatcaa tgtttactcg aacagaatgc atttcttcac 60tccgaagcca aatgacaaat aaagtccaaa ggcattttct cctgtgctga ccaaccaaat 120aatatgtata gacacacaca catatgcaca cacacacaca cacacccaca gagagagagc 180tgcaagagca tggaattcat gtgtttaaag ataatccttt ccatgtgaag tttaaaatta 240ctatatattt gctgatggct agattgagag aataaaagac agtaaccttt ctcttcaaag 300ataaaatgaa aagcaattgc tcttttcttc ctaaaaaatg caaaagattt acattgctgc 360caaatcattt caactgaaaa gaacagtatt gctttg 396 107 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 107 ttcacagaac anggtggtttattatttcaa tagcaaagag ctgaaaaatg tcgggtccca 60 taaaggagca gaacctgacccagagcctgc agtacatttc caccccacag gggtgcaggc 120 tgggccaggc agggccaaaggcagcagaaa tgggagtaag agactgtgcc cactgagaag 180 ctctgctggg tgtgggcaggtgggcatgan atgatgatga tgtagtgtaa ggaccaggta 240 ggcaaaacct gtcaggnttgntgaatgtca nagtggatcc aaaaggctga gggggtcgtc 300 anaaggccgg nggncccncccttgcccgta tgggccttca aaaagtatgc ttgctcatcc 360 gttgtttncc ccanggagctgccanggana aggctn 396 108 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 108 gcctgctttt gatgatgtct acagaaaatg ctggctgagc tgaacacatttgcccaattc 60 caggtgtgca cagaaaaccg agaatattca aaattccaaa tttttttcttaggagcaaga 120 agaaaatgtg gccctaaagg gggttagttg aggggtaggg ggtagtgaggatcttgattt 180 ggatctcttt ttatttaaat gtgaatttca acttttgaca atcaaagaaaagacttttgt 240 tgaaatagct ttactgcttc tcacgtgttt tggagaaaan natcanccctgcaatcactt 300 tttgnaactg ncnttgattt tcngcnncca agctatatcn aatatcgtctgngtanaaaa 360 tgncctggnc ttttgaanga atacatgngt gntgct 396 109 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 109 ggccgtaggcagccatggcg cccagcccgg aatggcatgg tcttgaagcc ccacttccac 60 aaggactggcagcggcgcgt ggccacgtgg ttcaaccagc cggcccggaa gatccgcaga 120 cgtaaggcccggcaagccaa ggcgcgccgc atcgctccgc gccccgcgtc gggtcccatc 180 cggcccatcgtgcgctgccc acggttcggt accacacgaa gggcgcgccg gcgcggnttc 240 agcctggaggagctcagggt ggccggattt acaagaagng gccngacatc ngtattcttg 300 ggatncnngaagnggaacaa gtcacngagt ccttgcagcc acntcagcgg ntgatgacac 360 cgttcnaactcatctnttcc caagaaacct cngnnc 396 110 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 110 nntgggctcc tnncantnat aataaaccngactcatacnc cacaaggaga tgaacaggan 60 tatgtncatn ctgacgcgga aacagngcanggagctgagg aggngccaag atgagaccta 120 nnggccnngg tgggcgcatt cccggnggagggggccacta aggantacga nnntcnagcg 180 gctcttgnng gcngncctcc tcacncctgnntattcgatt gtcncnnatg ncntcctatn 240 atnntcanna ttctntnntn atctcntntacnncntcncn ttcatgntta cngntccctc 300 tcnttctnac cnttntctgn anctcctttctnnnnctttc atctntnttc ngctttcttt 360 ctnnaatcnt nntttaacnt nntctnctttntnatt 396 111 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 111 taangancat nctggnttnt gcctnnccgn ctnattgant gttaaaggca attntgtggn60 tgtcccagng aatgncggct nattttcttt ccacattgng cncattcact cctcccactc 120ttggcatgtn gngacataag canggtacat aatngnaaaa atctgnattt ctgatgccan 180angggtanan cntnttgnat ntcattccat tgatatacag ccactntttt atttttgatc 240ancggccttc ggntcactgc ncanggtact tgacctcagt gtcactatta tgggntttgg 300tttcnctctt ttncnggccn ttntntttcn cacnttncan cttncttnnt nnaaaannna 360nncactctct cttgctctct ngatacnnng tctnaa 396 112 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 112 tcaacgtcac caattactgccatttagccc acgagctgcg tctcagctgc atggagagga 60 aaaaggtcca gattcgaagcatggatccct ccgccttggc aagcgaccga tttaacctca 120 tactggcaga taccaacagtgaccggctct tcacagtgaa cgatgttaaa gntggaggct 180 ccaagnatgg tatcatcaacctgcaaagtc tgaagacccc tacgctcaag gtgttcatgc 240 acgaaaacct ctacttcaccaaccggaagg tgaattcggg gggctgggcc tcgctgaatc 300 acttggattc cacattctgctatgcctcat gggactcgca gaacttcagg ctggccaccc 360 tgctcccacc atcactgntngncaatantc acccag 396 113 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 113 nnnnttnnnn nggagcctta atttcagagt tttattgtat tgcactaaaggaacagcagg 60 atggntatac aattttctct cattcagttt tgaaaatctg tagtacctgcaaattcttaa 120 gaataccttt accaccagat tagaacagta agcataataa ccaatttcttaataagtaat 180 gtcttacaaa taaaaacaca tttaaaatag ctttaaatgc attcttcacaagtaattcag 240 catatatttt atatcatggt tacttatgct tangaattnn agcaggatntttattctttt 300 gatggaaata tgggaaaact ntattcatgc atatacangg ataatattcagcgaagggaa 360 aatcccgttt ttattttggn aatgattcat atataa 396 114 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 114 aaatgggacaacgtgattct tttgttttaa ataaatactn agaacacgga cttggctcct 60 acaagcatttggactctaag gnttagaact ggagagtctt acccatgggc cccncncagg 120 gacgccacggttccctccca ccccgngatc aagacacgga atcngntggc gatngttgga 180 tcgcnatgtgccccttatct atagccttcc cnggncatnt acangcagga tgcggntggg 240 anaactacaactgnaatntc tcnaacggtn atggtcccca ccgatnaaga ttctacctng 300 tcttttcntcccctggagtg tgagtgnnng aggaagaagc ccttncctta catcaccttt 360 tgnacttctgaacaaganca anacnatggc cccccc 396 115 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 115 ccgcctggtt cggcccgcct gcctccactcctgcctctac catgtccatc agggtgaccc 60 agaagtccta caaggtgtcc acctctggcccccgggcctt cagcagccgc tcctacacga 120 gtgggcccgg ttcccgcatc agctcctcgagcttctcccg agtgggcagc agcaactttc 180 gcggtggcct ggcggcggct atggtggggccagcggcatg ggaggcatca cccgcagtta 240 cggcaaccag agcctgctga gccccttgcctggaggngga ccccaacatc aagccgngcg 300 cacccaggaa aaggagcaga ncaagaccctcaacaacaag nttgcttctt catagacaag 360 ggaccggtcc ttgaacagca naacaagatgntggag 396 116 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 116 atctcagttt actagctaag tgactttggg caagggattt aacctctcgt ccctcagttt60 cctcctatgt aaaatgacaa ggataatagt accaacccaa tgtagattaa atgagtttac 120gaagtgttag aatagtgctt ggcacattag tgctttacaa ctgctatttt gattgttgtt 180gtgggctctc tcaaatgcat tgtctctaga tgccagtgac ccaggtcaaa atttaccttt 240aaccaagctg catgtttccc agactgntgc acagtcctct accctgagan aaagcttcca 300cccaaggata cttttacttt ctgctggaaa actgatgagc aanggcaaca ngggacactt 360atcgccaact ggaaangaga aattcttcct tttgct 396 117 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 117 aaacattttt taataaaattcctatagaaa gctcagtcat agggcaaata ctcagttctc 60 tttcccatat caccgaggattgagagctcc caatattctt tggagaataa gcagtagttt 120 tgctggatgt tgccaggactcagagagatc acccatttac acattcaaac cagtagttcc 180 tattgcacat attaacattacttgccccta gcaccctaaa tatatggnac ctcaacaaat 240 aacttaaaga tttccgtggggcgcganacc atttcaattt gaactaatat ccttgaaaaa 300 aatcacatta ttacaagntttaataaatac nggaagaaga gctggcattt ttctaanatc 360 tgaattcnga cttggntttattccataaat acggtt 396 118 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 118 accnncacct gntnnntttt aacnattaca acttctttat atggcagtttttactgggng 60 cctaacactc tctttactgn ctcaagngga agtccaaaca aatttcatttttgtagtaaa 120 aaatctttat ttccaaaatg atttgttagc caaaagaact ataaaccacctaacaagact 180 ttggaagaaa gagacttgat gcttcttata aattccccat tgcanacaaaaaataacaat 240 ccaacaagag catggtaccc attcttacca ttaacctggn tttaannctccaaancnnga 300 tttaaaaatg accccactgg gcccaatcca acatganacc taggggggnttgccttgatt 360 angaatcccc cttanggact ttatctnggc tganaa 396 119 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 119 atggccagctcactttaaat accacctcaa gactcatcga aatgaccgct ccttcatctg 60 tcctgcagaaggttgtggga aaagcttcta tgtgctgcag aggctgaagg tgcacatgag 120 gacccacaatggagagaagc cctttatgtg ccatgagtct ggctgtggta agcagtttac 180 tacagctggaaacctgaaga accaccggcg catccacaca ggagagaaac ctttcctttg 240 tgaagcccaangatgtggcc gtcctttgct gagtattcta ncttcgaaaa catctggngg 300 ntactcangagagaaagcct cattantgcc antctgnggg aaaaccttct ntcagagngg 360 angcaggaatgtgcatatta aaaagctncc ttgnac 396 120 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 120 catgggtcag tcggtcctga gagttcgaagagggcacatt cccaaagaca ttcccagtca 60 tgaaatgtag aagactggaa aattaagacattatgtaaag gtagatatgg cttttagagt 120 tacattatgc ttggcatgaa taaggtgccaggaaaacagt ttaaaattat acatcagcat 180 acagactgct gttagaaggt atgggatcatattaagataa tctgcagctc tactacgcat 240 ttattgttaa ttgagttaca nangncattcannactgagt ttatagancc atattgctct 300 atctctgngn agaacatttg attccattgngaagaatgca gtttaaaata tctgaatgcc 360 atctagatgt attgtaccna aaggggaaaaataaca 396 121 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 121 tttttttttt ttttttttaa aatcaagtta tgtttaataa acattaataa atgtttactt60 aaaagggtta ataaacnttt actacatggc aaattatttt agctagaatg cttttggctt 120caagncatan aaaccagatt cnaatgccct taaanaattt tnaaanatcc attgangggg 180ataactgtaa tccccaaggg gaanagggtt gggtatgaca ggtacanggg gccagcccag 240tnntnncana nncagactct taccntcttt ctgctgtgnc accctcaggc attggctcca 300ttctcngggn tgcncatggg aagatggctt tggacntaac nacacccttt tgtncacgta 360aaggccngat gcagggtcaa anagnttccn ccatnt 396 122 396 DNA Homo sapien 122gtcgacatgg ctgccctctg ggctcccaga acccacaaca tgaaagaaat ggtgctaccc 60agctcaagcc tgggcctttg aatccggaca caaaaccctc tagcttggaa atgaatatgc 120tgcactttac aaccactgca ctacctgact caggaatcgg ctctggaagg tgaagctaga 180ggaaccagac ctcatcagcc caacatcaaa gacaccatcg gaacagcagc gcccgcagca 240cccaccccgc accggcgact ccatcttcat ggccaccccc tgcggtggac ggttgaccac 300cagccaccac atcatcccag agctgagctc ctccagcggg atgacgccgt ccccaccacc 360tccctcttct tctttttcat ccttctgtct ctttgt 396 123 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 123 gccctttttt tttttttttttttcctagtg ccaggtttat tccctcacat gggtggttca 60 catacacagc acanaggcacgggcaccatg gganagggca gcactcctgc cttctgaggg 120 gatcttggcc tcacggtgtaanaagggana ggatggtttc tcttctgccc tcactagggc 180 ctagggaacc cagnagcaaatcccaccacg ccttccatnt ctcagccaag ganaagccac 240 cttggtgacg tttagttccaaccattatag taagtggana agggattggc ctggtcccaa 300 ccattacagg gtgaanatataaacagtaaa ggaanataca gtttggatga ggccacagga 360 aggagcanat gacaccatcaaaagcatatg caggga 396 124 396 DNA Homo sapien 124 gaccattgcc ccagacctggaagatataac attcagttcc caccatctga ttaaaacaac 60 ttcctccctt acagagcatacaacagaggg ggcacccggg gaggagagca catactgtgt 120 tccaatttca cgcttttaattctcatttgt tctcacacca acagtgtgaa gtgcgtggta 180 taatctccat ttcaaaaccaaggaagcagc ctcagagtgg tcgagtgaca cacctcacgc 240 aggctgagtc cagagcttgtgctcctcttg attcctggtt tgactcagtt ccaggcctga 300 tcttgcctgt ctggctcagggtcaaagaca gaatggtgga gtgtagcctc cacctgatat 360 tcaggctact cattcagtcccaaatatgta ttttcc 396 125 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 125 cccttttttt tttttttttt tttttttttt ttttttactt tgnaacaaaaatttattagg 60 attaagtcaa attaaaaaac ttcatgcncc nccncttgtc atatttacctgaaatgacaa 120 agttatactt agcttgagng naaaacttgn gccccaaaaa ttntgtttggaaagcaaaaa 180 aataattgat gcncatagca gngggcctga tnccnccaca gngaatgttgtttaaggnct 240 aacaaacagg ggncancaaa gcatacatta cttttaagct ttgggnccaaggaaaangtc 300 attccctacc tccttcaaaa gcaaactcat natagcctgg gcncctaggnctggagcctn 360 ttttttcgag tctaanatga acatntggat ttcaan 396 126 396 DNAHomo sapien 126 cgcgtcgact cgcaagtgga atgtgacgtc cctggagacc ctgaaggctttgcttgaagt 60 caacaaaggg cacgaaatga gtcctcaggt ggccaccctg atcgaccgctttgtgaaggg 120 aaggggccag ctagacaaag acaccctaga caccctgacc gccttctaccctgggtacct 180 gtgctccctc agccccgagg agctgagctc cgtgcccccc agcagcatctgggcggtcag 240 gccccacgac ctggacacgc tggggctacg gctacagggc ggcatccccaacggctacct 300 ggtcctagac ctcagcatgc aagaggccct ctcggggacg ccctgcctcctaggacctgg 360 acctgttctc accgtcctgg cactgctcct agcctc 396 127 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 127 ttttttttttttggnggtaa aatgcaaatg ttttaaaata tgtttatttt gtatgtttta 60 caatgaatacttcagcaaag aaaataatta taatttcaaa atgcaatccc tggatttgat 120 aaatatcctttataatcgat tacactaatc aatatctaga aatatacata gacaaagtta 180 gctaatgaataaaataagta aaatgactac ataaactcaa tttcagggat gagggatcat 240 gcatgatcagttaagtcact ctgccacttt ttaaaataat acgattcaca tttgcttcaa 300 tcacataaacattcattgca ggagttacac ggctaatcat tgaaaattat gatctttgtt 360 agcttaaaagaaaattcagt ttaatacaaa gacatt 396 128 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 128 gccctttttt ttttttttta aaggcaaataaaataagttt attgggatgt aaccccatca 60 taaattgagg agcatccata caggcaagctataaaatctg gaaaatttaa atcaaattaa 120 attctgcttt taaaaaggtg ccttaagttaaccaagcatt ttgataacac attcaaattt 180 aatatataaa aatagatgta tcctggaagatataatgaan aacatgccat gtgtataaat 240 tcanaatacg ctttttacac aaagaactacaaaaagttac aaagacagcc ttcaggaacc 300 acacttagga aaagtgagcc gagcagccttcacgcaaagc ctccttcaaa naagtctcac 360 aaagactcca gaaccagccg agtntgtgaaaaagga 396 129 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 129 gccctttttt tttttttttt ttttactcag acaggcaata tttgctcaca tttattctct60 tgcatcgtaa atagtagcca actcacaaaa ataaagtata caanaatgta atatttttta 120aaataagatt aacagtgtaa gaaggaaaat ctcaaaaaaa gcanatagac aatgtanaaa 180attgaaatga aatcccacag taanaaaaaa aaaacanaaa agtgcctatt taanaattat 240gctacatgtg gaacttaact agaccatttt aanaaagacc aatttctaat gcaaattttc 300tgaggttttc anattttatt tttaaaatat gttatagcta catgttgtcn acncggccgc 360tcgagtctan agggcccgtt taaacccgct gatcag 396 130 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 130 cgcccttttt tttttttttttanngnacgt gnctttattt ctggatgata taaaanaaaa 60 aacttaaaaa acaccccaaaccaaacacca atggatcccc aaagcgatgt gactccctct 120 tcccacccgg ataaatagagacttctgtat gtcagtctac cctcccgccc ccataacccc 180 ctctgctata nacatactctgggtatatat tactctactc ggcaatagac atctcccgaa 240 aatagaattc ctgccctgacacctgactct tccctggccg catcanacca cccgccactg 300 tagcacactg gtgtccttgccccctgtggt cagggccatg ctgtcatccc acaanaaggc 360 cacatttgtc acatggctgctgtgtccacc gtactt 396 131 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 131 gccctttttt tttttttttt tttttttttt ttcagtttac acaaaaacnctttaattgac 60 agtatacnnt tttccaaaat atnttttngt aanaaaatgc aataattattaactatagtt 120 tttacaaaca agtttntcan taaattccag tgtncttnaa accccnnncnannaaaacat 180 atatganccc ccagttcctg ggcaaactgt tgaacattca ctgcanacaaaaagaccanc 240 nccaaanagt catctgngnc ctccatgctg ngtttgcacc aaacctgagggancagctag 300 ngaccgtgac aaaagctntg ctacagtttt actntngccc tntntgcctcccccatnatg 360 tttccttggt ccctcantcc tgtnggagta agttcc 396 132 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 132 cgcgtcgaccgcggccgtag cagccgggct ggtcctgctg cgagccggcg gcccggagtg 60 gggcggcgntatgtaccttc cacattgagt attcagaaag aagtgatctg aactctgacc 120 attctttatggatacattaa gtcaaatata agagtctgac tacttgacac actggctcgg 180 tgagttctgctttttctttt taatataaat ttattatgtt ggtaaattta gcttttggct 240 tttcactttgctctcatgat ataagaaaat gtaggttttc tctttcagtt tgaattttcc 300 tattcagtaaaacaacatgc tagaaaacaa acttttggaa aggcattgta actatttttt 360 caaatagaaccataataaca agtcttgtct taccct 396 133 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 133 ntattacccc tcctggnnan ntggnnatannctgcaaggn gatnnncccg nngaacttca 60 ctgatnnncc aatnaaaact gctttaaanctgactgcaca tatgaattnt aatacttact 120 tngcgggagg ggtggggcag ggacagcaagggggaggatt gggaanacaa tagacaggca 180 tgctggggat gcngcgggct ctatggcttctgangcgnaa agaaccagct ggggctctag 240 ggggtatccc cacgcgccct gtagcngcncattaaacgcg gcgggtgtgg nggttacttc 300 gcaaagngac cgatncactt gccagcgccctagctgcccg ctcctttngc tttcttccct 360 tcctttctcg ccacnttnnc cggctntccccgncaa 396 134 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 134 tttttttttt ttctgctttt tatatgttta aaaatctctc attctattgc tgctttattt60 aaagaaagat tactttcttc cctacaagat ctttattaat tgtaaaggga aaatgaataa 120ctttacaatg ganacacctg gcanacacca tcttaaccaa agcttgaagt taacataacc 180agtaatagaa ctgatcaata tcttgtgcct cctgatatgg ngtactaana aaaacacaac 240atcatgccat gatagtcttg ccaaaagtgc ataacctaaa tctaatcata aggaaacatt 300anacaaactc aaattgaagg acattctaca aagtgccctg tattaaggaa ttattcanag 360taaaggagac ttaaaagaca tggcaacaat gcagta 396 135 396 DNA Homo sapien 135gcgtcgacgc tggcagagcc acaccccaag tgcctgtgcc cagagggctt cagtcagctg 60ctcactcctc cagggcactt ttaggaaagg gtttttagct agtgtttttc ctcgctttta 120atgacctcag ccccgcctgc agtggctaga agccagcagg tgcccatgtg ctactgacaa 180gtgcctcagc ttccccccgg cccgggtcag gccgtgggag ccgctattat ctgcgttctc 240tgccaaagac tcgtgggggc catcacacct gccctgtgca gcggagccgg accaggctct 300tgtgtcctca ctcaggtttg cttcccctgt gcccactgct gtatgatctg ggggccacca 360ccctgtgccg gtggcctctg ggctgcctcc cgtggt 396 136 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 136 ttatgcttcc ggctcgtntgttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 60 acagctatga ccatgattacgccaagctat ttaggtgaca ctatagaata ctcaagctat 120 gcatcaagct tggtaccgagctcggatcca ctagtaacgg ccgccagtgt gctggaattc 180 gcggncgntc nantctagagggcccgttta aacccgctga tcagcctcga ctgtgccttc 240 tagttgccag ccatctgttgtttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 300 cactcccact gtcctttcctaataaaatga ggaaattgca tcgcattgtc tgagtaggtg 360 tcattctatt ctggggggtggggtggggca ggacan 396 137 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 137 tttttttttt ttctgctttg tacttgagtt tatttcacaa aaccacggagaaagatactg 60 aaatggagct ctttccagcc tccaagcaag gaggccccag cagccagtctccagcccctt 120 gagccctttt tgttaggccc acacccaaaa gagganaacc agtgtgtgcgcgaaggtaca 180 tggcaaggca cttttgaaaa catcccagtt taccgnggtg aaattgaacttactctgaaa 240 cagatgaaaa gggacatgca aaattgctga gcacatggag gtgtttgttagtaggtgaaa 300 atcatgtcct gggtataacc cagcttctcc aggttagggt gagccgccgtctggatcagt 360 ggtggcgggc cacacaccag gatgagcgtg gacttc 396 138 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 138 ccctttttttttttttttac aaatgagaaa aatgtttatt aagaaaacaa tttagcagct 60 ctcctttanaattttacaga ctaaagcaca acccgaaggc aattacagtt tcaatcatta 120 acacactacttaaggngctt gcttactcta caactggaaa gttgctgaag tttgtgacat 180 gccactgtaaatgtaagtat tattaaaaat tacaaattgt ttggtgatta ttttgatgac 240 ctcttgagcagcagctcccc ccaanaatgc ancaatggta tgtggctcac cagctccata 300 tcggcaaaattcgtggacat aatcatcttt caccattaca gataaaccat attcctgaag 360 gaagccagtgagacaagact tcaactttcc tatatc 396 139 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 139 ccgccctttt tttttttttt ttcacaaaagcactttttat ttgaggcaaa nagaagtctt 60 gctgaaagga ttccagttcc aagcagtcaaaactcaaccg ttagnggcac tattttgacc 120 tggtanattt tgcttctctt tggtcanaaaagggtattca ggttgtactt tccccagcag 180 ggtaaaaaga agggcaaagc aaactggaananacttctac tctactgaca gggctnttga 240 natccaacat caagctanac acnccctcgctggccactct acaggttgct gtcccactgc 300 tgagtgacac aggccatact acatttgcaaggaaaaaaat gaggcaanaa acacaggtat 360 aggtcacttg gggacgagca ggcaaccacagcttca 396 140 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 140 tttttttttt tttttttttt tttttttctc atttaacttt tttaatgggn ctcaaaattn60 tgngacaaat ttttggtcaa gttgtttcca ttaaaaagtn ctgattttaa aaactaataa 120cttaaaactg ccncncccaa aaaaaaaaac caaaggggtc cacaaaacat tntcctttcc 180ttntgaaggn tttacnatgc attgttatca ttaaccagtn ttttactact aaacttaaan 240ggccaattga aacaaacagt tntganaccg ttnttccncc actgattaaa agnggggggg 300caggtattag ggataatatt catttancct tntgagcttt ntgggcanac ttggngacct 360tgccagctcc agcagccttn ttgtccactg ntttga 396 141 396 DNA Homo sapien 141acgccgagcc acatcgctca gacaccatgg ggaaggtgaa ggtcggagtc aacggatttg 60gtcgtattgg gcgcctggtc accagggctg cttttaactc tggtaaagtg gatattgttg 120ccatcaatga ccccttcatt gacctcaact acatggttta catgttccaa tatgattcca 180cccatggcaa attccatggc accgtcaagg ctgagaacgg gaagcttgtc atcaatggaa 240atcccatcac catcttccag gagcgagatc cctccaaaat caagtggggc gatgctggcg 300ctgagtacgt cgtggagtcc actggcgtct tcaccaccat ggagaaggct ggggctcatt 360tgcagggggg agccaaaagg gtcatcatct ctgccc 396 142 396 DNA Homo sapien 142acgcaggaga ggaagcccag cctgttctac cagagaactt gcccaggtca gaggtctgcg 60tagaagccct tttctgagca tcctctcctc tcctcacacc tgccactgtc ctctgcgttg 120ctgtcgaatt aaatcttgca tcaccatggt gcacttctgt ggcctactca ccctccaccg 180ggagccagtg ccgctgaaga gtatctctgt gagcgtgaac atttacgagt ttgtggctgg 240tgtgtctgca actttgaact acgagaatga ggagaaagtt cctttggagg ccttctttgt 300gttccccatg gatgaagact ctgctgttta cagctttgag gccttggtgg atgggaagaa 360aattgtagca gaattacaag acaagatgaa ggcccg 396 143 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 143 tttttttttt tttccatanaaaataggatt tattttcaca tttaaggnga acacaaatcc 60 atgttccana aatgttttatgcataacaca tcatgagtag attgaatttc tttaacacac 120 anaaaaatca aagcctaccaggaaatgctt ccctccggag cacaggagct tacaggccac 180 ttntgttagc aacacaggaattcacattgt ctaggcacag ctcaagngag gtttgttccc 240 aggttcaact gctcctacccccatgggccc tcctcaaaaa cgacagcagc aaaccaacag 300 gcttcacagt aaccaggaggaaagatctca gngggggaac cttcacaaaa gccctgagtt 360 gtgtttcaaa agccaagctctggggtctgn ggcctg 396 144 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 144 tttttttttt tttcgctctt tggtctgaca agaaaagagt tttaggtgtgtgaagtaggg 60 tgggaaaaaa ggtcagtttc aaattcagta acatatggta acactaagttaggctgctgc 120 attcttttct ttgggtactt aagccagctg gcacttccac tttgtaaccaattatattat 180 gatcaacaac taatcagtta gttcctcagc ttcaactgaa nagttcctgattacctgatg 240 aaggacatac ttgctctggc ttcaattagc atgctgtcaa gcatccctctccatgcttaa 300 catggcaaca caaaacccaa gagtccttct ntttttttca ttagccatgaataaacactc 360 acaaagggga agagtagaca ctgcttttag taaacg 396 145 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 145 tttttttttttttttttcaa tggatccgtt agctttacta ctaanatctt gctganatca 60 nanaagggcttctgggcagg ctgagcactg ggggtgtgca acatggtaac tctgaataan 120 anaaaccctgagttttactg ggcaaanaaa naacaagngg taggtatgat ttctgaacct 180 ggaaatagcgaaaatgaagg aaattccaaa agcgcgtatt tccaaataat gacaggccag 240 caagaggacaccaaacctnt anaaagaggt attntttctt ccagctactg atggctttgg 300 catcccacaggcacattcct ttggccttca ggatcttana tgcanatgtg ganagtcaag 360 aggtaggctgactctgagtc ttcagctaaa ttcttt 396 146 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 146 tttttttttt ttttcattag caaggaaggatttatttttt cttttgaggg gagggcggaa 60 cagccgggat ttttggaaca ctacctttgtctttcacttt gttgtttgtg tgttaacacn 120 aataaatcan aagcgacttt aaatctcccttcgcaggact gtcttcacgt atcagngcan 180 acaanaaaac agtggcttta caaaaaanatgttcaagtag gctgcacttt gcctctgngg 240 gtgaggcaca ctgngggana nacaaggtcccctgnaacca gaggngggaa ggacanagct 300 ggctgactcc ctgctctccc gcattctctcctccatgtgt tttgaanagg gaagcaacat 360 gttgaggtct gatcatttct acccagggaacctgtt 396 147 396 DNA Homo sapien 147 acggggaagc caagtgaccg tagtctcatcagacatgagg gaatgggtgg ctccagagaa 60 agcagacatc attgtcagtg agcttctgggctcatttgct gacaatgaat tgtcgcctga 120 gtgcctggat ggagcccagc acttcctaaaagatgatggt gtgagcatcc ccggggagta 180 cacttccttt ctggctccca tctcttcctccaagctgtac aatgaggtcc gagcctgtag 240 ggagaaggac cgtgaccctg aggcccagtttgagatgcct tatgtggtac ggctgcacaa 300 cttccaccag ctctctgcac cccagccctgtttcaccttc agccatccca acagagatcc 360 tatgattgac aacaaccgct attgcaccttggaatt 396 148 396 DNA Homo sapien 148 acgtcccatg attgttccag accatgactcttcctggttg tgggtttgtt acagagcagg 60 agaagcagag gttatgacag ttatgcagactttccccctc ctttttctct tttctcttcc 120 ccttgctttt ccactgtttc ttcctgctgccacctgggcc ttgaattcct gggctgtgaa 180 gacatgtagc agctgcaggg tttaccacacgtgggagggc agcccagtac tgtccctctg 240 ccttccccac tttgagaata tggcagcccctttcattcct ggcttggggt aggggagacc 300 attgaagtag aagcctcaaa gcagacttttccctttactg tgtgtactcc aggacgaaga 360 aggaagatca tgcttgatac ttagattggttttccc 396 149 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 149 tttttttttt tttaaagagt cacattttat tcaatgccta tttgtacatg ttactagcaa60 taaactcttt tatctttaat tttgagaagt tttacaaata cagcaaagca gaatgactaa 120tagagccggt aaccaggaca cagatttgga aaaataggtc taattggttg ttacactgtg 180tttatgtcat acatttcgct tatttttatc aaanaaaaat cagaatttat aaaatgttaa 240ttaaaaggaa aacattctga gtaaatttag tcccgtgttt cttcctccaa atctntttgt 300tctacactaa caggtcagga taagtatgga tggggaggct ggaaaaaggg catccttccc 360catgcggtcc ccagagccac cctctccaag caggac 396 150 396 DNA Homo sapien 150acgcctctct tcagttggca cccaaacatc tggattggca aatcagtggc aagaagttcc 60agcatctgga cttttcagaa ttgatcttaa gtctactgtc atttccagat gcattatttt 120acaactgtat ccttggaaat atatttctag ggagaatatt attgaagaaa atgttaatag 180cctgagtcaa atttcagcag acttaccagc atttgtatca gtggtagcaa atgaagccaa 240actgtatctt gaaaaacctg ttgttccttt aaatatgatg ttgccacaag ctgcattgga 300gactcattgc agtaatattt ccaatgtgcc acctacaaga gagatacttc aagtctttct 360tactgatgta cacatgaagg aagtaattca gcagtt 396 151 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 151 acaaaatgcc cagcctacagagtctgagaa ggaaatttat aatcaggtga atgtagtatt 60 aaaagatgca gaaggcatcttggaggactt gcagtcatac agaggagctg gccacgaaat 120 acgagaggca atccagcatccagcanatga gaagttgcaa gagaaggcat ggggtgcagt 180 tgttccacta gtaggcaaattaaagaaatt ttacgaattt tctcagaggt tagaagcagc 240 attaagaggt cttctgggagccttaacaag taccccatat tctcccaccc agcatctana 300 gcgagagcag gctcttgctaaacagtttgc anaaattctt catttcacac tccggtttga 360 tgaactcaag atgacaaatcctgccataca gaatga 396 152 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 152 acgcagcgct cggcttcctg gtaattcttc acctcttttc tcagctccctgcagcatggg 60 tgctgggccc tccttgctgc tcgccgccct cctgctgctt ctctccggcgacggcgccgt 120 gcgctgcgac acacctgcca actgcaccta tcttgacctg ctgggcacctgggtcttcca 180 ggtgggctcc agcggttccc agcgcgatgt caactgctcg gttatgggaccacaagaaaa 240 aaaagtagng gtgtaccttc agaagctgga tacagcatat gatgaccttggcaattctgg 300 ccatttcacc atcatttaca accaaggctt tgagattgtg ttgaatgactacaagtggtt 360 tgcctttttt aagtataaag aagagggcag caaggt 396 153 396 DNAHomo sapien 153 ccagagacaa cttcgcggtg tggtgaactc tctgaggaaa aacacgtgcgtggcaacaag 60 tgactgagac ctagaaatcc aagcgttgga ggtcctgagg ccagcctaagtcgcttcaaa 120 atggaacgaa ggcgtttgcg gggttccatt cagagccgat acatcagcatgagtgtgtgg 180 acaagcccac ggagacttgt ggagctggca gggcagagcc tgctgaaggatgaggccctg 240 gccattgccg ccctggagtt gctgcccagg gagctcttcc cgccactcttcatggcagcc 300 tttgacggga gacacagcca gaccctgaag gcaatggtgc aggcctggcccttcacctgc 360 ctccctctgg gagtgctgat gaagggacaa catctt 396 154 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 154 acagcaaacctcctcacagc ccactggtcc tcaagagggg cnacntcttc acacatcanc 60 acaactacgcattgcctccc tncactcgga aggactatcc tgctgccaag agggtcaagt 120 tggacagtgtcagagtcctg agacagatca gcaacaaccg aaaatgcacc agccccaggt 180 cctcggacaccgaggagaat gtcaagaggc gaacacacaa cgtcttggag cgccagagga 240 ggaacgagctaaaacggagc ttttttgccc tgcgtgacca gatcccggag ttggaaaaca 300 atgaaaaggcccccaaggta gttatcctta aaaaagccac agcatacatc ctgtccgtcc 360 aagcagaggagcaaaagctc atttctgaag aggact 396 155 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 155 tttttttttt tgaananaca ggtctttaatgtacggagtc tcacaaggca caaacaccct 60 caccaggacc aaataaataa ctccacggttgcaggaaggc gcggtctggg gaggatgcgg 120 catctgagct ctcccagggc tggtgggcgagccgggggtc tgcagtctgt gaggggcctc 180 ctgggtgtgt ccgggcctct anagcgggtccagtctccag gatggggatc gctcactcac 240 tctccgagtc ggagtagtcc gccacgagggaggagccgan actgcagggg tgccgcgtgt 300 cgggggtgtc agctgcctcc tgggaggagcctgctggcna caggggcttg tcctgacggc 360 tcccttcctg ccccctcggg ctgctgcacttggggg 396 156 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 156 gaaggggggc ngggcagggg cggaatgtan anattantgc catgattgaa gatttaagaa60 acgtgagatt caggattttc accacatccc catttagtta gcttgctcgt ttggctggtg 120caaatgccag atggattatg aacaatgaca gtaaattaat gcaacataat caggtaatga 180tgccaagcgt atctggtgtt ccaggtattg tacctttacc ggaacaaatc agtaaatcca 240caatccctgg cacctgttag gcagctatta acctagtaaa tgctccccca tcccatctca 300atcagcaang acaatcaaaa acatttgctt tnagtggcag gaacactggt acatttttac 360ttgctccaag ggctgtgcca acgctccctc tctctg 396 157 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 157 tttttttttt tttttggggaatgtaaatct tttattaaaa cagttgtctt tccacagtag 60 taaagctttg gcacatacagtataaaaaat aatcacccac cataattata ccaaattcct 120 nttatcaact gcatactaagtgttttcaat acaatttttt ccgtataaaa atactgggaa 180 aaattgataa ataacaggtaananaaagat atttctaggc aattactagg atcatttgga 240 aaaagtgagt actgnggatatttaaaatat cacagtaaca agatcatgct tgttcctaca 300 gtattgcggg ccanacacttaagtgaaagc anaagtgttt gggtgacttt cctacttaaa 360 attttggnca tatcatttcaaaacatttgc atcttg 396 158 396 DNA Homo sapien 158 tttccgaaga cgggcagcttcagagaagag gattattcgg gagattgctg gtgtggccca 60 tagactcttt ggcatagactctttcgcagg cagccactct gagtgtggcc agttctataa 120 ccatccccaa actagctggagcctgatgga taggaacggg tagtctgtcc tcttccccat 180 aaaaatgttc caaaaagttatctccagaga gagtccctta tgaagacagt tgccaagctg 240 tattctcatt ctttaaaccaatacccaggt cagggctagt tcacactagc actgttaggg 300 acatggtgtg gctagaaatgaattgagtgt gacttctccc tacaacccca ggcccaggga 360 taggaggagg cagaggggtgcctggagttt ctgcac 396 159 396 DNA Homo sapien 159 tccgcgcgtt gggaggtgtagcgcggctct gaacgcgctg agggccgttg agtgtcgcag 60 gcggcgaggg cgcgagtgaggagcagaccc aggcatcgcg cgccgagaag gccgggcgtc 120 cccacactga aggtccggaaaggcgacttc cgggggcttt ggcacctggc ggaccctccc 180 ggagcgtcgg cacctgaacgcgaggcgctc cattgcgcgt gcgcgttgag gggcttcccg 240 cacctgatcg cgagaccccaacggctggtg gcgtcgcctg cgcgtctcgg ctgagctggc 300 catggcgcag ctgtgcgggctgaggcggag ccgggcgttt ctcgccctgc tgggatcgct 360 gctcctctct ggggtcctggcggccgaccg agaacg 396 160 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 160 ggaaaccttc tcaactaaga gaacatcatt tctggcaaac tatttttgttagctcacaat 60 atatgtcgta cactctacaa tgtaaatagc actganccac ancttacagaaggtaaaaag 120 angnataana acttccttta caaaanantt cctgttgttc ttaatactccccattgctta 180 tganaattnt ctatangtct ctcangantg ttcgcaccca tttcttttntaacttctact 240 aaaaanccat ttacattgna nagtgtacna cntatatttg ngagctaacaaaaaatngtt 300 ttccnganat gatgttcttt tagtttnaga nggttcnnnc aanttnctactccngcccgc 360 cactgnncnc cacatttnnn naattacacc ncacng 396 161 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 161 tttttgtttgattattttta ttataatgaa attaaactta tgactattac agtatgctca 60 gcttaaaacatttatgagta ctgcaaggac taacagaaac aggaaaaatc ctactaaaaa 120 tatttgttgatgggaaatca ttgtgaaagc aaacctccaa atattcattt gtaagccata 180 agaggataagcacaaccata tgggaggaga taaccagtct ctcccttcat atatattctt 240 ttttatttcttggtatacct tcccaaaaca nanacattca acagtagtta gaatggccat 300 ctcccaacattttaaaaaaa ctgcnccccc caatgggtga acaaagtaaa gagtagtaac 360 ctanagttcagctgagtaag ccactgtgga gcctta 396 162 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 162 tttttttttt tttttttttt ttttttttttttnggggncc aaattttttt ntttgaagga 60 angggacaaa nnaaaaaact taaggggntgttttggnncn acttanaaaa aagggaaagg 120 aaaccccaac atgcatgccc tnccttggggaccanggaan ncnccccncn ggtntgggga 180 aantaacccn aggnttaact ttnattatcactgncnccca gggggggctt nnaaaaaaaa 240 nnttccccca anccaaantn gggnncncccattttncnca anttggncnc cnggncnccc 300 nattttttga ngggtttcnc cngcncattnagggaanggg nntcaannaa accncncaaa 360 ngggggnnat ttttntcang ggccnatttgngcnnt 396 163 396 DNA Homo sapien 163 cactgtccgg ctctaacaca gctattaagtgctacctgcc tctcaggcac tctcctcgcc 60 cagtttctga ggtcagacga gtgtctgcgatgtcttcccg cactctattc ccccagcctc 120 tttctgcttt catgctcagc acatcatcttcctaggcagt ctcttcccca aagtctcacc 180 ttttcttcca atagaaaatt ccgcttgacctttggtgcac tgcccacttc ccagctccac 240 tggcccaagt ctgagccgga ggcccttgttttgggggcgg ggggagagtt ggatgtgatt 300 gcccttgaag aacaaggctg acctgagaggttcctggcgc cctgaggtgg ctcagcacct 360 gcccagggta ggcctggcat gaggggttaggtcagc 396 164 396 DNA Homo sapien 164 gacacgcggc ggtgtcctgt gttggccatggccgactacc tgattagtgg gggcacgtcc 60 tacgtgccag acgacggact cacagcacagcagctcttca actgcggaga cggcctcacc 120 tacaatgact ttctcattct ccctgggtacatcgacttca ctgcagacca ggtggacctg 180 acttctgctc tgaccaagaa aatcactcttaagaccccac tggtttcctc tcccatggac 240 acagtcacag aggctgggat ggccatagcaatggcgctta caggcggtat tggcttcatc 300 caccacaact gtacacctga attccaggccaatgaagttc ggaaagtgaa gaaatatgaa 360 cagggattca tcacagaccc tgtggtcctcagcccc 396 165 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 165 tttttttttt tttttttttt ttttttcang ggncactgag gctttttatt ttgancncaa60 aaccnccggg gatctancct gnggccnccc cggaaatnac ncnaggctca catnactnta 120aacncttggg ggaaagggag gcaaaaaaaa caatgacttg ggccaattnc ncnactgcaa 180agntananct gccaacaggg ctccagggag cttggnttnt gtaaaanttn taaggaagcg 240gnncnaactc cncggggggg gggcnctaac tancagggac ccctgcaagn gttggncggg 300ggcctcaacc tgcctgagct nacncaaggg gnggggtntn tntanccaac aggggaccna 360agggcttgcc tncccacagn ttacttggcc aagggg 396 166 396 DNA Homo sapienmisc_feature (1)...(396) n = A,T,C or G 166 ttttttcaaa ttcagagcatttttattaaa agaacaaaat attaaggcac aaaatacatc 60 aatttttcaa atgaaaacccttcaaacggt tatgtcctac attcaacgaa acttcttcca 120 aattacggaa taatttaactttttaaaata naaaaataca agttcttaaa tgcctaaaat 180 ttctccccaa ataaatgttttcttagtttt aatgaagtct cttcatgcag tactgagctc 240 caatattata atgtncacttccttaaaaat ctagttttgc cacttatata cattcaatat 300 gtttaaccag tatattaaccagtatattaa ccaatatgtt aaacttcttt taagtataag 360 gcttggtatt ttgtattgcttattgcatgc tttgat 396 167 396 DNA Homo sapien 167 tggcggcagc ggcggtggcggtggctgagc agaggacccg gcgggcggcc tcgcgggtca 60 ggacacaatg tttgcacgaggactgaagag gaaatgtgtt ggccacgagg aagacgtgga 120 gggagccctg gccggcttgaagacagtgtc ctcatacagc ctgcagcggc agtcgctcct 180 ggacatgtct ctggtgaagttgcagctttg ccacatgctt gtggagccca atctgtgccg 240 ctcagtcctc attgccaacacggtccggca gatccaagag gagatgacgc aggatgggac 300 gtggcgcaca gtggcaccccaggctgcaga gcgggcgccg ctcgaccgct tggtctccac 360 ggagatcctg tgccgtgcagcgtgggggca agaggg 396 168 396 DNA Homo sapien 168 taggatggta agagtattataaggattggt acaaggcatg atgagtcctt ttgcttttag 60 gcttttgact tctggttttagactttcttt agcttctgtt gttagacaac attgtgcaag 120 cttggttttt ataagtttgcatggattaaa ctgaacttaa tgaaattgtc cctcccccca 180 aattctcagc acaatttttaggcccacaag gagtcaagca cctcaaggag atcttcagtt 240 tgaacttggt gtagacacagggatactgat gaatcaatat tcaaattagc tgttacctac 300 ttaagaaaga gaggagaccttggggatttc gaggaagggt tcataaggga gattttagct 360 gagaaatacc atttgcacagtcaatcactt ctgacc 396 169 396 DNA Homo sapien misc_feature (1)...(396) n= A,T,C or G 169 tttttttttt tttcanaatt aaattcttta atacaaaatg cttttttttttttaaaanat 60 atctgtattt ctttgncgtt gttnaaaaat aaatatgtnc tacggaatatntcnaaaaac 120 tgcnctaaaa acaaanacgn gatgttaata tcttttcccc ncaattnttacggataaaca 180 gtanccccna taaataaatg atancnaatn ttaaaattaa aaaagganananatttagta 240 tgnaaaattc tctatttttt cttggtttgg ttttncntat aaaaaacanaatagcaatgt 300 ntnttttatc anaatcccnt ntntncctaa acnttttttt ttttntttncccccnaatnc 360 aagnngccaa anatntntnt agnatgnana tgtntn 396 170 396 DNAHomo sapien 170 tgagaagtac catgccgctt ctgcagagga acaggcaacc atcgaacgcaacccctacac 60 catcttccat caagcactga aaaactgtga gcctatgatt gggctggtacccatcctcaa 120 gggaggccgt ttctaccagg tccctgtacc cctacccgac cggcgtcgccgcttcctagc 180 catgaagtgg atgatcactg agtgccggga taaaaagcac cagcggacactgatgccgga 240 gaagctgtca cacaagctgc tggaggcttt ccataaccag ggccccgtgatcaagaggaa 300 gcatgacttg cacaagatgg cagaggccaa ccgtgccctg gcccactaccgctggtggta 360 gagtctccag gaggagccca gggccctctg cgcaag 396 171 396 DNAHomo sapien misc_feature (1)...(396) n = A,T,C or G 171 ggtcctcgtcgtggtgagcg cagccactca ggctggtcct gggggtgggg ctgtagggga 60 aagtgctaaagccgctgagt gaagtaagaa ctctgctaga gaggaaaatg ggcttgcttt 120 catcatcatcctnctcagct ggtggggtca agtgggaagt tctgtcactg ggatctggtt 180 cagtgtctcaagaccttgcc ccaccacgga aagccttttt cacntacccc aaaggacttg 240 gagagatgttagaagatggn tctnaaanat tcctctgcna atntgttttt agctatcaag 300 tggcttccccccttaancag gnaaaacatg atcagcangt tgctcggatg gaaaaactan 360 cttggtttgnnaaaaaanct ggaggcttga caatgg 396 172 396 DNA Homo sapien misc_feature(1)...(396) n = A,T,C or G 172 agccttgggc caccctcttg gagcatctggctgtcgaatt cttgtgaccc tgttacacac 60 actggagaga atgggcagaa gtcgtggtgttgcagccctg tgcattgggg gtgggatggg 120 aatagcaatg tgtgttcaga gagaatgaattgcttaaact ttgaacaacc tcaatttctt 180 tttaaactaa taaagtacta ggttgcaatatgtgaaaaaa aaaaaaaaag ggcggccgnt 240 cnantntana gggcccnttn aaacccgttgatcaacctcg actgtgcctt ctagttgcca 300 gccatctgtt gttngcccct cccccgtgnctttcttgacc ttgaaagggg ccccncccct 360 gtctttccta anaaaaanga agaantnnccttccnt 396 173 396 DNA Homo sapien misc_feature (1)...(396) n = A,T,C orG 173 aagcatgtgg atatgtttag ctacgtttac tcacagccag cgaactgaca ttaaaataac60 taacaaacag attcttttat gtgatgctgg aactcttgac agctataatt attattcaga 120aatgactttt tgaaagtaaa agcagcataa agaatttgtc acaggaaggc tgtctcagat 180aaattatggt aaaattttgc aggggacann ctttttaaga cttgcacaat tnccggatcc 240tgcnctgact ttggaaaagg catatatgtn ctagnggcat gganaatgcc ccatactcat 300gcatgcaaat taaacaacca agtttgaatc tttttggggg ngngctatnc tttaacccng 360tacnggcntt attatntaan gnccctgnnn cntgtg 396 174 924 DNA Homo sapiens 174ctgacgacc cggcgacggc gacgtctctt ttgactaaaa gacagtgtcc agtgctccag 60ctaggagtc tacggggacc gcctcccgcg ccgccaccat gcccaacttc tctggcaact 120gaaaatcat ccgatcggaa aacttcgagg aattgctcaa agtgctgggg gtgaatgtga 180gctgaggaa gattgctgtg gctgcagcgt ccaagccagc agtggagatc aaacaggagg 240agacacttt ctacatcaaa acctccacca ccgtgcgcac cacagagatt aacttcaagg 300tggggagga gtttgaggag cagactgtgg atgggaggcc ctgtaagagc ctggtgaaat 360ggagagtga gaataaaatg gtctgtgagc agaagctcct gaagggagag ggccccaaga 420ctcgtggac cagagaactg accaacgatg gggaactgat cctgaccatg acggcggatg 480cgttgtgtg caccagggtc tacgtccgag agtgagtggc cacaggtaga accgcggccg 540agcccacca ctggccatgc tcaccgccct gcttcactgc cccctccgtc ccaccccctc 600ttctaggat agcgctcccc ttaccccagt cacttctggg ggtcactggg atgcctcttg 660cagggtcttg ctttctttga cctcttctct cctcccctac accaacaaag aggaatggct 720gcaagagccc agatcaccca ttccgggttc actccccgcc tccccaagtc agcagtccta 780gccccaaacc agcccagagc agggtctctc taaaggggac ttgagggcct gagcaggaaa 840gactggccct ctagcttcta ccctttgtcc ctgtagccta tacagtttag aatatttatt 900tgttaatttt attaaaatgc ttta 924 175 3321 DNA Homo sapiens 175 tgaagattttgatacttgg tatttttctg tttttatgta gtaccccagc ctgggcgaaa 60 aaaagcattattacattgg aattattgaa acgacttggg attatgcctc tgaccatggg 120 aaaagaaacttatttctgt tgacacggaa cattccaata tctatcttca aaatggccca 180 atagaattgggagactata taagaaggcc ctttatcttc agtacacaga tgaaaccttt 240 aggacaactatagaaaaacc ggtctggctt gggtttttag gccctattat caaagctgaa 300 actggagataaagtttatgt acacttaaaa aaccttgcct ctaggcccta cacctttcat 360 tcacatggaataacttacta taaggaacat gagggggcca tctaccctga taacaccaca 420 gattttcaaagagcagatga caaagtatat ccaggagagc agtatacata catgttgctt 480 gccactgaagaacaaagtcc tggggaagga gatggcaatt gtgtgactag gatttaccat 540 tcccacattgatgctccaaa agatattgcc tcaggactca tcggaccttt aataatctgt 600 aaaaaagattctctagataa agaaaaagaa aaacatattg accgagaatt tgtggtgatg 660 ttttctgtggtggatgaaaa tttcagctgg tacctagaag acaacattaa aacctactgc 720 tcagaaccagagaaagttga caaagacaac gaagacttcc aggagagtaa cagaatgtat 780 tctgtgaatggatacacttt tggaagtctc ccaggactct ccatgtgtgc tgaagacaga 840 gtaaaatggtacctttttgg tatgggtaat gaagttgatg tgcacgcagc tttctttcac 900 gggcaagcactgactaacaa gaactaccgt attgacacaa tcaacctctt tcctgctacc 960 ctgtttgatgcttatatggt ggcccagaac cctggagaat ggatgctcag ctgtcagaat 1020 ctaaaccatctgaaagccgg tttgcaagcc tttttccagg tccaggagtg taacaagtct 1080 tcatcaaaggataatatccg tgggaagcat gttagacact actacattgc cgctgaggaa 1140 atcatctggaactatgctcc ctctggtata gacatcttca ctaaagaaaa cttaacagca 1200 cctggaagtgactcagcggt gttttttgaa caaggtacca caagaattgg aggctcttat 1260 aaaaagctggtttatcgtga gtacacagat gcctccttca caaatcgaaa ggagagaggc 1320 cctgaagaagagcatcttgg catcctgggt cctgtcattt gggcagaggt gggagacacc 1380 atcagagtaaccttccataa caaaggagca tatcccctca gtattgagcc gattggggtg 1440 agattcaataagaacaacga gggcacatac tattccccaa attacaaccc ccagagcaga 1500 agtgtgcctccttcagcctc ccatgtggca cccacagaaa cattcaccta tgaatggact 1560 gtccccaaagaagtaggacc cactaatgca gatcctgtgt gtctagctaa gatgtattat 1620 tctgctgtggatcccactaa agatatattc actgggctta ttgggccaat gaaaatatgc 1680 aagaaaggaagtttacatgc aaatgggaga cagaaagatg tagacaagga attctatttg 1740 tttcctacagtatttgatga gaatgagagt ttactcctgg aagataatat tagaatgttt 1800 acaactgcacctgatcaggt ggataaggaa gatgaagact ttcaggaatc taataaaatg 1860 cactccatgaatggattcat gtatgggaat cagccgggtc tcactatgtg caaaggagat 1920 tcggtcgtgtggtacttatt cagcgccgga aatgaggccg atgtacatgg aatatacttt 1980 tcaggaaacacatatctgtg gagaggagaa cggagagaca cagcaaacct cttccctcaa 2040 acaagtcttacgctccacat gtggcctgac acagagggga cttttaatgt tgaatgcctt 2100 acaactgatcattacacagg cggcatgaag caaaaatata ctgtgaacca atgcaggcgg 2160 cagtctgaggattccacctt ctacctggga gagaggacat actatatcgc agcagtggag 2220 gtggaatgggattattcccc acaaagggag tgggaaaagg agctgcatca tttacaagag 2280 cagaatgtttcaaatgcatt tttagataag ggagagtttt acataggctc aaagtacaag 2340 aaagttgtgtatcggcagta tactgatagc acattccgtg ttccagtgga gagaaaagct 2400 gaagaagaacatctgggaat tctaggtcca caacttcatg cagatgttgg agacaaagtc 2460 aaaattatctttaaaaacat ggccacaagg ccctactcaa tacatgccca tggggtacaa 2520 acagagagttctacagttac tccaacatta ccaggtgaaa ctctcactta cgtatggaaa 2580 atcccagaaagatctggagc tggaacagag gattctgctt gtattccatg ggcttattat 2640 tcaactgtggatcaagttaa ggacctctac agtggattaa ttggccccct gattgtttgt 2700 cgaagaccttacttgaaagt attcaatccc agaaggaagc tggaatttgc ccttctgttt 2760 ctagtttttgatgagaatga atcttggtac ttagatgaca acatcaaaac atactctgat 2820 caccccgagaaagtaaacaa agatgatgag gaattcatag aaagcaataa aatgcatgct 2880 attaatggaagaatgtttgg aaacctacaa ggcctcacaa tgcacgtggg agatgaagtc 2940 aactggtatctgatgggaat gggcaatgaa atagacttac acactgtaca ttttcacggc 3000 catagcttccaatacaagca caggggagtt tatagttctg atgtctttga cattttccct 3060 ggaacataccaaaccctaga aatgtttcca agaacacctg gaatttggtt actccactgc 3120 catgtgaccgaccacattca tgctggaatg gaaaccactt acaccgttct acaaaatgaa 3180 gacaccaaatctggctgaat gaaataaatt ggtgataagt ggaaaaaaga gaaaaaccaa 3240 tgattcataacaatgtatgt gaaagtgtaa aatagaatgt tactttggaa tgactataaa 3300 cattaaaagagactggagca t 3321 176 487 DNA Homo sapiens 176 gaaatacttt ctgtcttattaaaattaata aattattggt ctttacaaga cttggataca 60 ttacagcaga catggaaatataattttaaa aaatttctct ccaacctcct tcaaattcag 120 tcaccactgt tatattaccttctccaggaa ccctccagtg gggaaggctg cgatattaga 180 tttccttgta tgcaaagtttttgttgaaag ctgtgctcag aggaggtgag aggagaggaa 240 ggagaaaact gcatcataactttacagaat tgaatctaga gtcttccccg aaaagcccag 300 aaacttctct gcagtatctggcttgtccat ctggtctaag gtggctgctt cttccccagc 360 catgagtcag tttgtgcccatgaataatac acgacctgtt atttccatga ctgctttact 420 gtatttttaa ggtcaatatactgtacattt gataataaaa taatattctc ccaaaaaaaa 480 aaaaaaa 487 177 3999 DNAHomo sapiens 177 caagattcca catttgatgg ggtgactgac aaacccatct tagactgctgtgcctgcgga 60 actgccaagt acagactcac attttatggg aattggtccg agaagacacacccaaaggat 120 taccctcgtc gggccaacca ctggtctgcg atcatcggag gatcccactccaagaattat 180 gtactgtggg aatatggagg atatgccagc gaaggcgtca aacaagttgcagaattgggc 240 tcacccgtga aaatggagga agaaattcga caacagagtg atgaggtcctcaccgtcatc 300 aaagccaaag cccaatggcc agcctggcag cctctcaacg tgagagcagcaccttcagct 360 gaattttccg tggacagaac gcgccattta atgtccttcc tgaccatgatgggccctagt 420 cccgactgga acgtaggctt atctgcagaa gatctgtgca ccaaggaatgtggctgggtc 480 cagaaggtgg tgcaagacct gattccctgg gacgctggca ccgacagcggggtgacctat 540 gagtcaccca acaaacccac cattccccag gagaaaatcc ggcccctgaccagcctggac 600 catcctcaga gtcctttcta tgacccagag ggtgggtcca tcactcaagtagccagagtt 660 gtcatcgaga gaatcgcacg gaagggtgaa caatgcaata ttgtacctgacaatgtcgat 720 gatattgtag ctgacctggc tccagaagag aaagatgaag atgacacccctgaaacctgc 780 atctactcca actggtcccc atggtccgcc tgcagctcct ccacctgtgacaaaggcaag 840 aggatgcgac agcgcatgct gaaagcacag ctggacctca gcgtcccctgccctgacacc 900 caggacttcc agccctgcat gggccctggc tgcagtgacg aagacggctccacctgcacc 960 atgtccgagt ggatcacctg gtcgccctgc agcatctcct gcggcatgggcatgaggtcc 1020 cgggagaggt atgtgaagca gttcccggag gacggctccg tgtgcacgctgcccactgag 1080 gaaacggaga agtgcacggt caacgaggag tgctctccca gcagctgcctgatgaccgag 1140 tggggcgagt gggacgagtg cagcgccacc tgcggcatgg gcatgaagaagcggcaccgc 1200 atgatcaaga tgaaccccgc agatggctcc atgtgcaaag ccgagacatcacaggcagag 1260 aagtgcatga tgccagagtg ccacaccatc ccatgcttgc tgtccccatggtccgagtgg 1320 agtgactgca gcgtgacctg cgggaagggc atgcgaaccc gacagcggatgctcaagtct 1380 ctggcagaac ttggagactg caatgaggat ctggagcagg tggagaagtgcatgctccct 1440 gaatgcccca ttgactgtga gctcaccgag tggtcccagt ggtcggaatgtaacaagtca 1500 tgtgggaaag gccacgtgat tcgaacccgg atgatccaaa tggagcctcagtttggaggt 1560 gcaccctgcc cagagactgt gcagcgaaaa aagtgccgca tccgaaaatgccttcgaaat 1620 ccatccatcc aaaagctacg ctggagggag gcccgagaga gccggcggagtgagcagctg 1680 aaggaagagt ctgaagggga gcagttccca ggttgtagga tgcgcccatggacggcctgg 1740 tcagaatgca ccaaactgtg cggaggtgga attcaggaac gttacatgactgtaaagaag 1800 agattcaaaa gctcccagtt taccagctgc aaagacaaga aggagatcagagcatgcaat 1860 gttcatcctt gttagcaagg gtacgagttc cccagggctg cactctagattccagagtca 1920 ccaatggctg gattatttgc ttgtttaaga caatttaaat tgtgtacgctagttttcatt 1980 tttgcagtgt ggttcgccca gtagtcttgt ggatgccaga gacatcctttctgaatactt 2040 cttgatgggt acaggctgag tggggcgccc tcacctccag ccagcctcttcctgcagagg 2100 agtagtgtca gccaccttgt actaagctga aacatgtccc tctggagcttccacctggcc 2160 agggaggacg gagactttga cctactccac atggagaggc aaccatgtctggaagtgact 2220 atgcctgagt cccagggtgc ggcaggtagg aaacattcac agatgaagacagcagattcc 2280 ccacattctc atctttggcc tgttcaatga aaccattgtt tgcccatctcttcttagtgg 2340 aactttaggt ctcttttcaa gtctcctcag tcatcaatag ttcctggggaaaaacagagc 2400 tggtagactt gaagaggagc attgatgttg ggtggctttt gttctttcactgagaaattc 2460 ggaatacatt tgtctcaccc ctgatattgg ttcctgatgc ccccccaacaaaaataaata 2520 aataaattat ggctgcttta tttaaatata aggtagctag tttttacacctgagataaat 2580 aataagctta gagtgtattt ttcccttgct tttgggggtt cagaggagtatgtacaattc 2640 ttctgggaag ccagccttct gaactttttg gtactaaatc cttattggaaccaagacaaa 2700 ggaagcaaaa ttggtctctt tagagaccaa tttgcctaaa ttttaaaatcttcctacaca 2760 catctagacg ttcaagtttg caaatcagtt tttagcaaga aaacatttttgctatacaaa 2820 cattttgcta agtctgccca aagccccccc aatgcattcc ttcaacaaaatacaatctct 2880 gtactttaaa gttattttag tcatgaaatt ttatatgcag agagaaaaagttaccgagac 2940 agaaaacaaa tctaagggaa aggaatatta tgggattaag ctgagcaagcaattctggtg 3000 gaaagtcaaa cctgtcagtg ctccacacca gggctgtggt cctcccagacatgcatagga 3060 atggccacag gtttacactg ccttcccagc aattataagc acaccagattcagggagact 3120 gaccaccaag ggatagtgta aaaggacatt ttctcagttg ggtccatcagcagtttttct 3180 tcctgcattt attgttgaaa actattgttt catttcttct tttataggccttattactgc 3240 ttaatccaaa tgtgtaccat tggtgagaca catacaatgc tctgaatacactacgaattt 3300 gtattaaaca catcagaata tttccaaata caacatagta tagtcctgaatatgtacttt 3360 taacacaaga gagactattc aataaaaact cactgggtct ttcatgtctttaagctaagt 3420 aagtgttcag aaggttcttt tttatattgt cctccacctc catcattttcaataaaagat 3480 agggcttttg ctcccttgtt cttggaggga ccattattac atctctgaactacctttgta 3540 tccaacatgt tttaaatcct taaatgaatt gctttctccc aaaaaaagcacaatataaag 3600 aaacacaaga tttaattatt tttctacttg gggggaaaaa agtcctcatgtagaagcacc 3660 cacttttgca atgttgttct aagctatcta tctaactctc agcccatgataaagttcctt 3720 aagctggtga ttcctaatca aggacaagcc accctagtgt ctcatgtttgtatttggtcc 3780 cagttgggta cattttaaaa tcctgatttt ggagacttaa aaccaggttaatggctaaga 3840 atgggtaaca tgactcttgt tggattgtta ttttttgttt gcaatggggaatttataaga 3900 agcatcaagt ctctttctta ccaaagtctt gttaggtggt ttatagttcttttggctaac 3960 aaatcatttt ggaaataaag attttttact acaaaaatg 3999 178 1069DNA Homo sapiens 178 aaaaaagatg aataaatgaa taagagagat gaataaacaaatttacatta catgtgatag 60 ttatcatggt atggccttca tgacaagatg gatgagaatatcactgatag gatattagcc 120 ttctttcata tctttatatt gaaatatggg ctttacttcaatttgaaggt ctttcatgaa 180 caataaaaga gagtagaagg actgtctgag aaggcaggagacatataaaa cagatgactg 240 aaagactgac tagctcctgg aaagggaaac atttggaacatccagagtaa gggcaaatgg 300 gcttctacca gcacaacaaa gagcctccag gtggcaacatggaagcaggt tatcagagaa 360 aataaatgtg caaattcctt atttacaatg actcacttaaccccacaaac atgtttcact 420 gctgccttcc ccagttgtcg cttatgtact gttgttacctttcagttaca tgcctttgat 480 cctaaaattc tctacttttg gtgccttatc agttctttgcaatctgcctg tggttatcag 540 cacttaaagc acaattttga aggggaaaaa aatgataatcaccttagtcc caaagaaata 600 atttgtcaaa ctgccttatt agtattaaaa acagacacactgaatgaagt agcatgatac 660 gcatatatcc tactcagtat cattggcctt ttatcaaatggggaaactat acttttgtat 720 tacatagttt tagaaatcga aagttagaga ctctttataagtaatgtcaa ggaacagtaa 780 tttaaaaaca aagttctaac aaatatattg tttgcttaatcacaatgccc tcaacttgta 840 tttgaataac taaataggac atgtcttcct tggagctgtgggcattagtt cagaagcact 900 acctgcatct taattttcaa aacttaagtt ttattagcaaatcctcttct ctgtaagact 960 tagctatgaa gtggtatatt ttttccaaat atttttctgaaaacatttgt tgttgtaact 1020 gcacaataaa agtccagttg caattaaaaa aaaaaaaaaaaaaaaaaaa 1069 179 1817 DNA Homo sapiens 179 tgctattctg ccaaaagacaatttctagag tagttttgaa tgggttgatt tcccccactc 60 ccacaaactc tgaagccagtgtctagctta ctaaaaaaag agttgtatat aatatttaag 120 atgctgagta tttcataggaaagctgaatg ctgctgtaaa gtgctcttta agtctttttt 180 ttttttaatc cccttctaatgaatgaaact aggggaattt caggggacag agatgggatt 240 tgttgtatga taaactgtatgtagttttta gtctttctgt tttgagaagc agtggttggg 300 gcatttttaa gatggctggctactcttgtt ttccctcatg ataataaatt tgtcataact 360 cagtaacatg aacttgcccctagaggtagt tgttaataat tttgaaatat taaggtcttg 420 ccaagcttct gatgattcacacctgtacta ctgattatta agcaggacag actgagcttt 480 ctgttgcaaa taccttggaggagaaagtaa tttctaaata tacagagagg taacttgact 540 atatatgttg catcctgtgcctcccttcat attaatattt gataaagatt ttaatttatg 600 taaaacttct aaagcagaatcaaagctcct cttggggaaa tggcaagtct ttaggatagg 660 caagaccctg tatgaatagtaccaaagcat taccgcatgg tagagaacac actcgattaa 720 aaatgttaag ctatctgaaaaataaaatgt gcaagtcttc aggatggcac aaaacaaagg 780 ttaatgcttc ttggggcacatttcttagag ggcttgctga gtgtgtaaat ataatcgact 840 tttgtttgtg ttacatgacttctgtgactt cattgaaaat ctgcacaatt cagtttcagc 900 tctggattac ttcagttgacctttgtgaag gtttttatct gtgtagaatg ggtgtttgac 960 ttgttttagc ctattaaatttttattttct ttcactctgt attaaaagta aaacttacta 1020 aaagaaaaga ggtttgtgttcacattaaat ggttttggtt tggcttcttt tagtcaggct 1080 ttctgaacat tgagatatcctgaacttaga gctcttcaat cctaagattt tcatgaaaag 1140 cctctcactt gaacccaaaccagagtactc ttactgcctc ttttctaaat gttcaggaaa 1200 agcattgcca gttcagtcttttcaaaatga gggagaaaca tttgcctgcc ttgtaataac 1260 aagactcagt gcttattttttaaactgcat tttaaaaatt ggatagtata ataacaataa 1320 ggagtaagcc accttttataggcaccctgt agttttatag ttcttaatct aaacatttta 1380 tatttccttc ttttggaaaaaacctacatg ctacaagcca ccatatgcac agactataca 1440 gtgagttgag ttggctctcccacagtcttt gaggtgaatt acaaaagtcc agccattatc 1500 atcctcctga gttatttgaaatgatttttt ttgtacattt tggctgcagt attggtggta 1560 gaatatacta taatatggatcatctctact tctgtattta tttatttatt actagacctc 1620 aaccacagtc ttctttttccccttccacct ctctttgcct gtaggatgta ctgtatgtag 1680 tcatgcactt tgtattaatatattagaaat ctacagatct gttttgtact ttttatactg 1740 ttggatactt ataatcaaaacttttactag ggtattgaat aaatctagtc ttactagaaa 1800 aaaaaaaaaa aaaaaaa 1817180 2382 DNA Homo sapiens 180 acttttattg gaagcagcag ccacatccctgcatgatttg cattgcaata caaccataac 60 cgggcagcca ctcctgagtg ataaccagtataacataaac gtagcagcct caatttttgc 120 ctttatgacg acagcttgtt atggttgcagtttgggtctg gctttacgaa gatggcgacc 180 gtaacactcc ttagaaactg gcagtcgtatgttagtttca cttgtctact ttatatgtct 240 gatcaatttg gataccattt tgtccagatgcaaaaacatt ccaaaagtaa tgtgtttagt 300 agagagagac tctaagctca agttctggtttatttcatgg atggaatgtt aattttatta 360 tgatattaaa gaaatggcct tttattttacatctctcccc tttttccctt tcccccttta 420 ttttcctcct tttctttctg aaagtttccttttatgtcca taaaatacaa atatattgtt 480 cataaaaaat tagtatccct tttgtttggttgctgagtca cctgaacctt aattttaatt 540 ggtaattaca gcccctaaaa aaaacacatttcaaataggc ttcccactaa actctatatt 600 ttagtgtaaa ccaggaattg gcacactttttttagaatgg gccagatggt aaatatttat 660 gcttcacggt ccatacagtc tctgtcacaactattcagtt ctgctagtat agcgtgaaag 720 cagctataca caatacagaa atgaatgagtgtggttatgt tctaataaaa cttatttata 780 aaaacaaggg gaggctgggt ttagcctgtgggccatagtt tgtcaaccac tggtgtaaaa 840 ccttagttat atatgatctg cattttcttgaactgatcat tgaaaactta taaacctaac 900 agaaaagcca cataatattt agtgtcattatgcaataatc acattgcctt tgtgttaata 960 gtcaaatact tacctttgga gaatacttacctttggagga atgtataaaa tttctcaggc 1020 agagtcctgg atataggaaa aagtaatttatgaagtaaac ttcagttgct taatcaaact 1080 aatgatagtc taacaactga gcaagatcctcatctgagag tgcttaaaat gggatcccca 1140 gagaccatta accaatactg gaactggtatctagctactg atgtcttact ttgagtttat 1200 ttatgcttca gaatacagtt gtttgccctgtgcatgaata tacccatatt tgtgtgtgga 1260 tatgtgaagc ttttccaaat agagctctcagaagaattaa gtttttactt ctaattattt 1320 tgcattactt tgagttaaat ttgaatagagtattaaatat aaagttgtag attcttatgt 1380 gtttttgtat tagcccagac atctgtaatgtttttgcact ggtgacagac aaaatctgtt 1440 ttaaaatcat atccagcaca aaaactatttctggctgaat agcacagaaa agtattttaa 1500 cctacctgta gagatcctcg tcatggaaaggtgccaaact gttttgaatg gaaggacaag 1560 taagagtgag gccacagttc ccaccacacgagggcttttg tattgttcta ctttttcagc 1620 cctttacttt ctggctgaag catccccttggagtgccatg tataagttgg gctattagag 1680 ttcatggaac atagaacaac catgaatgagtggcatgatc cgtgcttaat gatcaagtgt 1740 tacttatcta ataatcctct agaaagaaccctgttagatc ttggtttgtg ataaaaatat 1800 aaagacagaa gacatgagga aaaacaaaaggtttgaggaa atcaggcata tgactttata 1860 cttaacatca gatcttttct ataatatcctactactttgg ttttcctagc tccataccac 1920 acacctaaac ctgtattatg aattacatattacaaagtca taaatgtgcc atatggatat 1980 acagtacatt ctagttggaa tcgtttactctgctagaatt taggtgtgag attttttgtt 2040 tcccaggtat agcaggctta tgtttggtggcattaaattg gtttctttaa aatgctttgg 2100 tggcactttt gtaaacagat tgcttctagattgttacaaa ccaagcctaa gacacatctg 2160 tgaatactta gatttgtagc ttaatcacattctagacttg tgagttgaat gacaaagcag 2220 ttgaacaaaa attatggcat ttaagaatttaacatgtctt agctgtaaaa atgagaaagt 2280 gttggttggt tttaaaatct ggtaactccatgatgaaaag aaatttattt tatacgtgtt 2340 atgtctctaa taaagtattc atttgataaaaaaaaaaaaa aa 2382 181 2377 DNA Homo sapiens 181 atctttatgc aagacaagagtcagccatca gacactgaaa tatattatga tagattatga 60 agaattttct ctgtagaattatattcttcc tggaacctgg tagagtagat tagactcaaa 120 ggctttttct tccttttcttactcctgttt tttccactca ctcttcccaa gagatttcct 180 aaagcttcaa gcttaataagcctaatagtg aaaaataact gaatttaatg gtataatgaa 240 gttcttcatt tccagacatctttaattgat cttaaagctc atttgagtct ttgcccctga 300 acaaagacag acccattaaaatctaagaat tctaaatttt cacaactgtt tgagcttctt 360 ttcattttga aggatttggaatatatatgt tttcataaaa gtatcaagtg aaatatagtt 420 acatgggagc tcaatcatgtgcagattgca ttctgttatg ttgactcaat atttaattta 480 caactatcct tatttatattgacctcaaga actccatttt atgcaatgca gaccactgag 540 atatagctaa cattctttcaaataattttc cttttctttt ataattcctc tatagcaaat 600 ttttatgtat aactgattatacatatccat atttatattt cattgattcc aagacatcac 660 tttttcaatt taacatctctgaaattgtga catttcttgc aactgttggc acttcagatg 720 cagtgtttaa aattatgcttgaataaatat tacactaatc caactttacc taaatgttta 780 tgcatctagg caaattttgttttcttataa agatttgaga gcccatttat gacaaaatat 840 gaaggcgaaa tttaaggacaactgagtcac gcacaactca acatggagcc taactgatta 900 tcagctcaga tcccgcatatcttgagttta caaaagctct ttcaggtccc catttatact 960 ttacgtgagt gcgaatgatttcagcaaacc ctaacttaac taacaagaat gggtaggtat 1020 gtctacgttt cattaacaaatttttattat ttttattcta ttatatgaga tccttttata 1080 ttatcatctc acttttaaacaaaattaact ggaaaaatat tacatggaac tgtcatagtt 1140 aggttttgca gcatcttacatgtcttgtat caatggcagg agaaaaatat gataaaaaca 1200 atcagtgctg tgaaaaacaactttcttcta gagtcctctt actttttatt cttctttatc 1260 atttgtgggt ttttcccccttggctctcac tttaacttca agcttatgta acgactgtta 1320 taaaactgca tatttaaattatttgaatta tatgaaataa ttgttcagct atctgggcag 1380 ctgttaatgt aaacctgagagtaataacac tactctttta tctacctgga atacttttct 1440 gcataaaatt tatctttgtaagctaactct attaatcagg tttcttctag cctctgcaac 1500 ctacttcagt tagaattgtctaatactgct ctattaatca ggtttctacc ctctacaacc 1560 tacttcagtt aaaattgtctaatacagcaa tatttaaaaa aaaaacactg caattgtcaa 1620 ggatggaaaa tgtgtgatttgtgtaaacaa tttttaccaa ctttacattt tcctacagat 1680 aaatgtgaaa ttttgataagaagtctacgc aatgacaagt acggtacata aattttatta 1740 agaatattga gtataaagtactttaattct aaattataag aaaatataca tttgcacata 1800 ttaatataga aattcattttgtgtatattt aacatagctt ttaaactatt ttacattagc 1860 tacttcatta tggtttcttgaacttctgaa aaaaattaga aatgtattaa acttatcagt 1920 aacataaaaa cttattttgtttcacctaac gaatactgcg tttgtaaaaa taaatttaat 1980 atagaatata tttttaaattaaatatttga atataaaata gctctaagaa agaagcaaat 2040 tatcactgaa catatttcttattatttctg gctttgaatt atacgtaact taaattgtct 2100 taaatgatac agaatattggagaatatgat actttcacat aatatactat gaacctgttc 2160 atataactct gattgactactaacttctgt tttatgtatt tattaaagag ctgacactgt 2220 agtttgtggt gagatgtttatttttctaac agagcttata acagttagga caaggcattt 2280 aattaatgca tcattctgtttagtagtagg tgttaatcaa tatgaaattc tctgttttaa 2340 aataaaaatg taaaaatctaaaaaaaaaaa aaaaaaa 2377 182 1370 DNA Homo sapiens 182 tgtgagcatggtattttgtc tcggaagaaa aaaatatggg tcaggcgcaa agtaagccca 60 ccccactgggaactatgtta aaaaaaaatt tcaagattta agggagatta cggtgttact 120 atgacaccagaaaaacttag aactttgtgt gaaatagact ggctaacatt agaggtgggt 180 tggctatcagaagaaagcct ggagaggtcc cttgtttcaa aggtatggca caaggtaacc 240 tgtaagccaaagcacccgga ccagtttcta tacatagaca gttacagctg gtttagaccc 300 cttccccctctccccacagt agttaagaga acagcagcat aagcagctgg cagaggcaag 360 gaaagaccagcagagagaaa aaaaggccat ctataccaat tttaagttaa tttagactga 420 acaagggcttattaatagca aaggataatt gaaatcacaa acttataagg gtttcaacaa 480 aagtgaagtttgctaaaagt taacagtgta acatgtatta tggtaacttc taatcttgtg 540 gccttagacagtctagtcaa aacacataaa gaaagtttgc tttaaaaaaa caatggttat 600 cttcaaaaataaaggggaga ggcagaattt atataaaaag agttatatga taaattcttg 660 tcctgaaataaattaactgg ttgtttaaag aaaagaatgt ttgtaataag tcaaaaagtt 720 aaaacatgtttaaaaaattg tctgcaaaag tcataaaaga aaaaatttta ttaaaaaaat 780 tttaagcaaaaaatgttgta taatttaaaa gtaataaggc ctcctgtgta ctattaagac 840 agatgcaaattcctggttga aatggatcaa atattccatc tgcacattaa acaaaagcaa 900 ttgttatgcttgtgcacatg gcaggccaga ggccctgatt gtcccccttc cactaaggtg 960 gtcctctagtcgaccaggcg tggactgcat ggtagctctt ttccaggatt ctacagcctg 1020 gagtaataagtcatgccaag ctctctctgc tatatcccaa agtctctgcg ggtcagcccc 1080 caagggccatgcagcttctg tctcccaaca ctaagttcac ttcgtgtctc tcacggcaga 1140 gaggaaacttagtattcctt ggagacctga agggatgcag tgagcttaag aattttcaag 1200 agcttatcaatcagtcagcc cttgttcatc cccgagtgga tgtgtggtgg tattgtggtg 1260 gacctttactgggcactctg ccaaataact agtgtggcac ttgtgcttta gtccatttgg 1320 ctatccctttcaccctggca tttcatcaac caaaaaaaaa aaaaaaaaaa 1370 183 2060 DNA Homosapiens misc_feature (1)...(2060) n=A,T,C or G 183 gtttcagggg aggagacaaggtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60 gtgaccgtgg aggccatctggccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120 gaagaccttt gagcaagaaagtaccctgga acaacccaat ttggactgca agtattagtt 180 gggtcttcca ggtgcctctcacagcagcag tcatggcagc agtgactcta gccatgtcca 240 tgaccaactg ctgcataacaaatagccccg agactcagca gcttacaaca gggtccccag 300 cccacagact ggcactggtccatggcttgt taggaacctg actgcgcagc agaaggtgag 360 tgagcattac tgcctgagctctgcctcctg tcagatcatc aggggcatta gattctcata 420 ggagcgtgaa ccctattgcaaaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480 agaatctaac taatgcctgatgatttgagg tggggcagtt tcatccccaa accatctctc 540 tcccttcatg tccatggaaaaattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600 tggagactgc tggtttacaaccgcaatgaa cattcatcat cccacacagt gtcagagggt 660 cgggaacacg ggtgccctgcctgtgtgctt ccggttccag atttctcagt gggttgtgat 720 caaggtatca gcggaggccgtattcatctg caagcttgac caggaataga agagccactt 780 catgggtggc tcactcagatgccagcaggt cagtgctggt ggctggcagg cagcctcagc 840 tcctcacctc atggatctctcctgagcaca gttttcctgt ccttacaacc tggtagctgg 900 cttctccaga gcaggtgactcaggagagga caaggtgaga gcccagcacc ttatggtcta 960 gtctcagaag tcacacgccatcatttctgc aatgtcattt tggggttcca ggtcagctgt 1020 atcactgtgg gaggtgagtatatagatgtc ctagaccatt caggctgcta tgacagaaca 1080 ccatgaactg agtggctcatgaacaacaga aatttcccac agttctgtag gctgggaaat 1140 ccaagatcaa ggtggcagcaggttcagcgt ctgctaagct cctgcttttc atggattgca 1200 tcttctcact gtgtcctcacgtgatggaca gagcaaatga gctctcaggc actagtccca 1260 gccatgagga ctctgctttcatgactcatc actccgcaaa ggcccacctc catcagaaga 1320 cagctgctaa ctgcagctgccatcctccaa gacgggagac acagaattgg gggacatata 1380 cattgagatc tgaaaggcctggacagcaac aggtggggat cgtgggggca tcttggaggg 1440 tggctgccgc agtaacatttctgacccatg ctttctgctt gcactcatct cctgcctttg 1500 atcttcatta tctcargcagtccccacaac gactgtatct aggagttcat tttaccctca 1560 ttttacagat gaaacgtctcagagggtaat gtgcttgccc agtgtctcac aaatgcaaag 1620 tcactgaggt aggatttcaacctaggtcca atcatctctg cagcattagg ggttcaccat 1680 tgccatagac ttaactgtgtcccccaaaat ttgtatgttg aagccctacc agcctccccc 1740 ccccaatgtg ctgatgtttggagaaagggc ctttgggagg taattaggtt tagatgagat 1800 catgagggtg ggactctcataatggcatta atgccatcag gtgaagagat accagagacc 1860 ttgtgtcctc tctctctgcaatgtgaggac acagtgagaa ggcagctgtc tgcaagctgg 1920 gaagagagta ctgaccaggaacttaatcag agggcatctt gatcttggac ttcccagcct 1980 ccagaactct gaaaagttaatgnctattat ttaagccacg cagtctatgg aattttgtta 2040 gagccaaccc caagcttact2060 184 3079 DNA Homo sapiens 184 ggcacaaagt tgggggccgc gaagatgaggctgtccccgg cgcccctgaa gctgagccgg 60 actccggcac tgctggccct ggcgctgcccctggccgcgg cgctggcctt ctccgacgag 120 accctggaca aagtgcccaa gtcagagggctactgtagcc gtatcctgcg cgcccagggc 180 acgcggcgcg agggctacac cgagttcagcctccgcgtgg agggcgaccc cgacttctac 240 aagccgggaa ccagctaccg cgtaacactttcagctgctc ctccctccta cttcagagga 300 ttcacattaa ttgccctcag agagaacagagagggtgata aggaagaaga ccatgctggg 360 accttccaga tcatagacga agaagaaactcagtttatga gcaattgccc tgttgcagtc 420 actgaaagca ctccacggag gaggacccggatccaggtgt tttggatagc accaccagcg 480 ggaacaggct gcgtgattct gaaggccagcatcgtacaaa aacgcattat ttattttcaa 540 gatgagggct ctctgaccaa gaaactttgtgaacaagatt ccacatttga tggggtgact 600 gacaaaccca tcttagactg ctgtgcctgcggaactgcca agtacagact cacattttat 660 gggaattggt ccgagaagac acacccaaaggattaccctc gtcgggccaa ccactggtct 720 gcgatcatcg gaggatccca ctccaagaattatgtactgt gggaatatgg aggatatgcc 780 agcgaaggcg tcaaacaagt tgcagaattgggctcacccg tgaaaatgga ggaagaaatt 840 cgacaacaga gtgatgaggt cctcaccgtcatcaaagcca aagcccaatg gccagcctgg 900 cagcctctca acgtgagagc agcaccttcagctgaatttt ccgtggacag aacgcgccat 960 ttaatgtcct tcctgaccat gatgggccctagtcccgact ggaacgtagg cttatctgca 1020 gaagatctgt gcaccaagga atgtggctgggtccagaagg tggtgcaaga cctgattccc 1080 tgggacgctg gcaccgacag cggggtgacctatgagtcac ccaacaaacc caccattccc 1140 caggagaaaa tccggcccct gaccagcctggaccatcctc agagtccttt ctatgaccca 1200 gagggtgggt ccatcactca agtagccagagttgtcatcg agagaatcgc acggaagggt 1260 gaacaatgca atattgtacc tgacaatgtcgatgatattg tagctgacct ggctccagaa 1320 gagaaagatg aagatgacac ccctgaaacctgcatctact ccaactggtc cccatggtcc 1380 gcctgcagct cctccacctg tgacaaaggcaagaggatgc gacagcgcat gctgaaagca 1440 cagctggacc tcagcgtccc ctgccctgacacccaggact tccagccctg catgggccct 1500 ggctgcagtg acgaagacgg ctccacctgcaccatgtccg agtggatcac ctggtcgccc 1560 tgcagcatct cctgcggcat gggcatgaggtcccgggaga ggtatgtgaa gcagttcccg 1620 gaggacggct ccgtgtgcac gctgcccactgaggaaatgg agaagtgcac ggtcaacgag 1680 gagtgctctc ccagcagctg cctgatgaccgagtggggcg agtgggacga gtgcagcgcc 1740 acctgcggca tgggcatgaa gaagcggcaccgcatgatca agatgaaccc cgcagatggc 1800 tccatgtgca aagccgagac atcacaggcagagaagtgca tgatgccaga gtgccacacc 1860 atcccatgct tgctgtcccc atggtccgagtggagtgact gcagcgtgac ctgcgggaag 1920 ggcatgcgaa cccgacagcg gatgctcaagtctctggcag aacttggaga ctgcaatgag 1980 gatctggagc aggtggagaa gtgcatgctccctgaatgcc ccattgactg tgagctcacc 2040 gagtggtccc agtggtcgga atgtaacaagtcatgtggga aaggccacgt gattcgaacc 2100 cggatgatcc aaatggagcc tcagtttggaggtgcaccct gcccagagac tgtgcagcga 2160 aaaaagtgcc gcatccgaaa atgccttcgaaatccatcca tccaaaagcc acgctggagg 2220 gaggcccgag agagccggcg gagtgagcagctgaaggaag agtctgaagg ggagcagttc 2280 ccaggttgta ggatgcgccc atggacggcctggtcagaat gcaccaaact gtgcggaggt 2340 ggaattcagg aacgttacat gactgtaaagaagagattca aaagctccca gtttaccagc 2400 tgcaaagaca agaaggagat cagagcatgcaatgttcatc cttgttagca agggtacgag 2460 ttccccaggg ctgcactcta gattccagagtcaccaatgg ctggattatt tgcttgttta 2520 agacaattta aattgtgtac gctagttttcatttttgcag tgtggttcgc ccagtagtct 2580 tgtggatgcc agagacatcc tttctgaatacttcttgatg ggtacaggct gagtggggcg 2640 ccctcacctc cagccagcct cttcctgcagaggagtagtg tcagccacct tgtactaagc 2700 tgaaacatgt ccctctggag cttccacctggccagggagg acggagactt tgacctactc 2760 cacatggaga ggcaaccatg tctggaagtgactatgcctg agtcccaggg tgcggcaggt 2820 aggaaacatt cacagatgaa gacagcagattccccacatt ctcatctttg gcctgttcaa 2880 tgaaaccatt gtttgcccat ctcttcttagtggaacttta ggtctctttt caagtctcct 2940 cagtcatcaa tagttcctgg ggaaaaacagagctggtaga cttgaagagg agcattgatg 3000 ttgggtggct tttgttcttt cactgagaaattcggaatac atttgtctca cccctgatat 3060 tggttcctga tgccccagc 3079 185 3000DNA Homo sapiens 185 gtttcagggg aggagacaag gtttcttgtt tgccgtatatgctcctgcag agaagaggaa 60 gtgaccgtgg aggccatctg gccctgtgtt ttgatatggcaaaattaatg aatgcaatca 120 gaagaccttt gagcaagaaa gtaccctgga acaacccaatttggactgca agtattagtt 180 gggtcttcca ggtgcctctc acagcagcag tcatggcagcagtgactcta gccatgtcca 240 tgaccaactg ctgcataaca aatagccccg agactcagcagcttacaaca gggtccccag 300 cccacagact ggcactggtc catggcttgt taggaacctgactgcgcagc agaaggtgag 360 tgagcattac tgcctgagct ctgcctcctg tcagatcatcaggggcatta gattctcata 420 ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatgtacgttgcgt gctccttatg 480 agaatctaac taatgcctga tgatttgagg tggggcagtttcatccccaa accatctctc 540 tcccttcatg tccatggaaa aattgtcttc tacaaaaccagtccgtggtg ccaaaaaggt 600 tggagactgc tggtttacaa ccgcaatgaa cattcatcatcccacacagt gtcagagggt 660 cgggaacacg ggtgccctgc ctgtgtgctt ccggttccagatttctcagt gggttgtgat 720 caaggtatca gcggaggccg tattcatctg caagcttgaccaggaataga agagccactt 780 catgggtggc tcactcagat gccagcaggt cagtgctggtggctggcagg cagcctcagc 840 tcctcacctc atggatctct cctgagcaca gttttcctgtccttacaacc tggtagctgg 900 cttctccaga gcaggtgact caggagagga caaggtgagagccacagcac cttatggtct 960 agtctcagaa gtcacacgcc atcatttctg caatgtcattttggggttcc aggtcagctg 1020 tatcactgtg ggaggtgagt atatagatgt cctagaccattcaggctgct atgacagaac 1080 accatgaact gagtggctca tgaacaacag aaatttcccacagttctgta ggctgggaaa 1140 tccaagatca aggtggcagc aggttcagcg tctgctaagctcctgctttt catggattgc 1200 atcttctcac tgtgtcctca cgtgatggac agagcaaatgagctctcagg cactagtccc 1260 agccatgagg actctgcttt catgactcat cactccgcaaaggcccacct ccatcagaag 1320 acagctgcta actgcagctg ccatcctcca agacgggagacacagaattg ggggacatat 1380 acattgagat ctgaaaggcc tggacagcaa caggtggggatcgtgggggc atcttggagg 1440 gtggctgccg cagtaacatt tctgacccat gctttctgcttgcactcatc tcctgccttt 1500 gatcttcatt atctcaggca gtccccacaa cgactgtatctaggagttca ttttaccctc 1560 attttacaga tgaaacgtct cagagggtaa tgtgcttgcccagtgtctca caaatgcaaa 1620 gtcactgagg taggatttca acctaggtcc aatcatctctgcagcattag gggttcacca 1680 ttgccataga cttaactgtg tcccccaaaa tttgtatgttgaagccctac cagcctcccc 1740 cccccaatgt gctgatgttt ggagaaaggg cctttgggaggtaattaggt ttagatgaga 1800 tcatgagggt gggactctca taatggcatt aatgccatcaggtgaagaga taccagagac 1860 cttgtgtcct ctctctctgc aatgtgagga cacagtgagaaggcagctgt ctgcaagctg 1920 ggaagagagt actgaccagg aacttaatca gagggcatcttgatcttgga cttcccagcc 1980 tccagaactc tgaaaagtta atgtctatta tttaagccacgcagtctatg gaattttgtt 2040 agagccaacc caagcttact aagataatca gtatgctgcactttctataa atgtaatttt 2100 tacatttata aaaacaaaac aagagatttg ctgctctataacaactgtac ctacattgta 2160 gatggaataa caaatctaca tacagattta gtaatctctatgtagatata gaacatagtg 2220 tatctaatag agacatagtg tctgtggtct gatgttaattttaggaatta gccgtcactg 2280 attgggcctt gtccaggtat tcttctccct tgtcctggctctgtaaccta gttatccttg 2340 tctttgctaa cccataacca actattgtat caggactattatgccactac agatgatgca 2400 gtttgggttt actgtttctc accatttaga caatacttcatcaaatatat ttctgtatga 2460 ctttagtgat atcagttttt gattcattcc tgcatagatctgggcaaatt gtagacctta 2520 ggaggtgtat tcaccatcca gttctctgga actgcttatgacatttttct ctgagctttc 2580 ttgtcccaaa aggagccttc ctaaaatagt ctttaagtgcctttaaaaag agaaagagaa 2640 attaagagaa aaaaaacccc aaactcattc ctttactctgatgtgacagt cctcccagga 2700 cactgcagtg gcctgagttt tgctgttaat ttcattcacttatgtttggg ctatgtaaat 2760 tctgcctaga gctggaatgt cattatgtaa agaaatattttttgtttata ttctttaata 2820 gtaccagtaa tgtatatctt attcagcttc gagaatataattgggttgtt tataaaaacc 2880 acacatcatc aaactcacat tgtaacgatt atttcacttttcaaaaaaaa tggcattaga 2940 aaaacttgaa tgatgttagt tatcttaaag aagtgtgtactatgtttaaa aaaaaaaaaa 3000 186 807 PRT Homo sapiens 186 Met Arg Leu SerPro Ala Pro Leu Lys Leu Ser Arg Thr Pro Ala Leu 5 10 15 Leu Ala Leu AlaLeu Pro Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu 20 25 30 Thr Leu Asp LysVal Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu 35 40 45 Arg Ala Gln GlyThr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg 50 55 60 Val Glu Gly AspPro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val 65 70 75 80 Thr Leu SerAla Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile 85 90 95 Ala Leu ArgGlu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly 100 105 110 Thr PheGln Ile Ile Asp Glu Glu Glu Thr Gln Phe Met Ser Asn Cys 115 120 125 ProVal Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg Ile Gln 130 135 140Val Phe Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Ile Leu Lys 145 150155 160 Ala Ser Ile Val Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser165 170 175 Leu Thr Lys Lys Leu Cys Glu Gln Asp Ser Thr Phe Asp Gly ValThr 180 185 190 Asp Lys Pro Ile Leu Asp Cys Cys Ala Cys Gly Thr Ala LysTyr Arg 195 200 205 Leu Thr Phe Tyr Gly Asn Trp Ser Glu Lys Thr His ProLys Asp Tyr 210 215 220 Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile GlyGly Ser His Ser 225 230 235 240 Lys Asn Tyr Val Leu Trp Glu Tyr Gly GlyTyr Ala Ser Glu Gly Val 245 250 255 Lys Gln Val Ala Glu Leu Gly Ser ProVal Lys Met Glu Glu Glu Ile 260 265 270 Arg Gln Gln Ser Asp Glu Val LeuThr Val Ile Lys Ala Lys Ala Gln 275 280 285 Trp Pro Ala Trp Gln Pro LeuAsn Val Arg Ala Ala Pro Ser Ala Glu 290 295 300 Phe Ser Val Asp Arg ThrArg His Leu Met Ser Phe Leu Thr Met Met 305 310 315 320 Gly Pro Ser ProAsp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys 325 330 335 Thr Lys GluCys Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro 340 345 350 Trp AspAla Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys 355 360 365 ProThr Ile Pro Gln Glu Lys Ile Arg Pro Leu Thr Ser Leu Asp His 370 375 380Pro Gln Ser Pro Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gln Val 385 390395 400 Ala Arg Val Val Ile Glu Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn405 410 415 Ile Val Pro Asp Asn Val Asp Asp Ile Val Ala Asp Leu Ala ProGlu 420 425 430 Glu Lys Asp Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr SerAsn Trp 435 440 445 Ser Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Asp LysGly Lys Arg 450 455 460 Met Arg Gln Arg Met Leu Lys Ala Gln Leu Asp LeuSer Val Pro Cys 465 470 475 480 Pro Asp Thr Gln Asp Phe Gln Pro Cys MetGly Pro Gly Cys Ser Asp 485 490 495 Glu Asp Gly Ser Thr Cys Thr Met SerGlu Trp Ile Thr Trp Ser Pro 500 505 510 Cys Ser Ile Ser Cys Gly Met GlyMet Arg Ser Arg Glu Arg Tyr Val 515 520 525 Lys Gln Phe Pro Glu Asp GlySer Val Cys Thr Leu Pro Thr Glu Glu 530 535 540 Met Glu Lys Cys Thr ValAsn Glu Glu Cys Ser Pro Ser Ser Cys Leu 545 550 555 560 Met Thr Glu TrpGly Glu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met 565 570 575 Gly Met LysLys Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly 580 585 590 Ser MetCys Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met Met Pro 595 600 605 GluCys His Thr Ile Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser 610 615 620Asp Cys Ser Val Thr Cys Gly Lys Gly Met Arg Thr Arg Gln Arg Met 625 630635 640 Leu Lys Ser Leu Ala Glu Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln645 650 655 Val Glu Lys Cys Met Leu Pro Glu Cys Pro Ile Asp Cys Glu LeuThr 660 665 670 Glu Trp Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys Gly LysGly His 675 680 685 Val Ile Arg Thr Arg Met Ile Gln Met Glu Pro Gln PheGly Gly Ala 690 695 700 Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys ArgIle Arg Lys Cys 705 710 715 720 Leu Arg Asn Pro Ser Ile Gln Lys Pro ArgTrp Arg Glu Ala Arg Glu 725 730 735 Ser Arg Arg Ser Glu Gln Leu Lys GluGlu Ser Glu Gly Glu Gln Phe 740 745 750 Pro Gly Cys Arg Met Arg Pro TrpThr Ala Trp Ser Glu Cys Thr Lys 755 760 765 Leu Cys Gly Gly Gly Ile GlnGlu Arg Tyr Met Thr Val Lys Lys Arg 770 775 780 Phe Lys Ser Ser Gln PheThr Ser Cys Lys Asp Lys Lys Glu Ile Arg 785 790 795 800 Ala Cys Asn ValHis Pro Cys 805 187 892 DNA Homo sapiens 187 tttattgatg tttcaacaggcacttattca aataagttat atatttgaaa acagccatgg 60 taagcatcct tggcttctcacccattcctc atgtggcatg ctttctagac tttaaaatga 120 ggtaccctga atagcactaagtgctctgta agctcaagga atctgtgcag tgctacaaag 180 cccacaggca gagaaagaactcctcaagtg cttgtggtca gagactaggt tccatatgag 240 gcacacctat gatgaaggtcttcacctcca gaaggtgaca ctgttcagag atcctcattt 300 cctggagagt gggagaaaatccctcctttg ggaaatccct tttcccagca gcagagccca 360 cctcattgct tagtgatcatttggaaggca ctgagagcct tcaggggctg acagcagaga 420 aatgaaaatg agtacagttcagatggtgga agaagcatgg cagtgacatc ttccatgctc 480 tttttctcag tgtctgcaactccaaagatc aaggccataa cccaggagac catcaacgga 540 agattagttc tttgtcaagtgaatgaaatc caaaagcacg catgagacca atgaaagttt 600 ccgcctgttg taaaatctattttcccccaa ggaaagtcct tgcacagaca ccagtgagtg 660 agttctaaaa gatacccttggaattatcag actcagaaac ttttattttt tttttctgta 720 acagtctcac cagacttctcataatgctct taatatattg cacttttcta atcaaagtgc 780 gagtttatga gggtaaagctctactttcct actgcagcct tcagattctc atcattttgc 840 atctattttg tagccaataaaactccgcac tagcaaaaaa aaaaaaaaaa aa 892 188 1448 DNA Homo sapiensmisc_feature (1)...(1448) n = A,T,C or G 188 tgtgactcac atttcttttactgtgacaca ataatgtgat cctaaaactg gcttatcctt 60 gagtgtttac aactcaaacaactttttgaa tgcagtagtt tttttttttt aaaaacaaac 120 ttttatgtca aattttttttcttagaagta gtcttcatta ttataaattt gtacaccaaa 180 aggccatggg gaactttgtgcaagtacctc atcgctgagc aaatggagct tgctatgttt 240 taatttcaga aaatttcctcatatacgtag tgtgtagaat caagtctttt aataattcat 300 tttttcttca taatatttactcaaagttaa gcttaaaaat aagttttatc ttaaaatcat 360 atttgaagac agtaagacagtaaactattt taggaagtca acccccattg cactctgtgg 420 cagttattct ggtaaaaataggcaaaagtg acctgaatct acaatggtgt cccaaagtaa 480 ccaagtaaga gagattgtaaatgataaacc gagctttaaa ggataaagtg ttaataaaga 540 aaggaagctg ggcacatgtcaaaaagggag atcgaaatgt taggtaatca tttagaaagg 600 acagaaaata tttaaagtggctcataggta atgaatattt ctgacttaga tgtaaatcca 660 tctggaatct ttacatcctttgccagctga aacaagaaag tgaagggaca atgatatttc 720 atggtcagtt tattttgtaagagacagaag aaattatatc tatacattac cttgtagcag 780 cagtacctgg aagccccagcccgtcacaga agtgtggagg ggggctcctg actagacaat 840 ttccctagcc cttgtgatttgaagcatgaa agttctggca ggttatgagc agcactaggg 900 ataaagtatg gttttattttggtgtaattt aggtttttca acaaagccct tgtctaaaat 960 aaaaggcatt attggaaatatttgaaaact agaaaatgat ggataaaagg gctgataaga 1020 aaatttctga ctgtcagtagaagtgagata agatcctcag aggaaacagt aagaagggat 1080 aatcattaag atagtaaaacaggcaaagca gaatcacatg tgcncacaca catacacatg 1140 taaacattgg aatgcataagttttaatatt ttagcgctat cagtttctaa atgcattaat 1200 tactaactgc cctctcccaagattcattta gttcaaacag tatccgtaaa ctaggaataa 1260 tgccacatgc attcaatgggatcttttaag tactcttcag tttgttccaa gaaatgtgcc 1320 tactgaaatc aaattaatttgtattcaatg tgtacttcaa gactgctaat tgtttcatct 1380 gaaagcctac aatgaatcattgttcamcct tgaaaaataa aattttgtaa atcaaaaaaa 1440 aaaaaaaa 1448 189 460DNA Homo sapiens 189 ttttgggagc acggactgtc agttctctgg gaagtggtcagcgcatcctg cagggcttct 60 cctcctctgt cttttggaga accagggctc ttctcaggggctctagggac tgccaggctg 120 tttcagccag gaaggccaaa atcaagagtg agatgtagaaagttgtaaaa tagaaaaagt 180 ggagttggtg aatcggttgt tctttcctca catttggatgattgtcataa ggtttttagc 240 atgttcctcc ttttcttcac cctccccttt tttcttctattaatcaagag aaacttcaaa 300 gttaatggga tggtcggatc tcacaggctg agaactcgttcacctccaag catttcatga 360 aaaagctgct tcttattaat catacaaact ctcaccatgatgtgaagagt ttcacaaatc 420 cttcaaaata aaaagtaatg acttaaaaaa aaaaaaaaaa460 190 481 DNA Homo sapiens 190 aggtggtgga agaaactgtg gcacgaggtgactgaggtat ctgtgggagc taatcctgtc 60 caggtggaag taggagaatt tgatgatggtgcagaggaaa ccgaagagga ggtggtggcg 120 gaaaatccct gccagaacca ccactgcaaacacggcaagg tgtgcgagct ggatgagaac 180 aacaccccca tgtgcgtgtg ccaggaccccaccagctgcc cagcccccat tggcgagttt 240 gagaaggtgt gcagcaatga caacaagaccttcgactctt cctgccactt ctttgccaca 300 aagtgcaccc tggagggcac caagaagggccacaagctcc acctggacta catcgggcct 360 tgcaaataca tccccccttg cctggactctgagctgaccg aattccccct gcgcatgcgg 420 gactggctca agaacgtcct ggtcaccctgtatgagaggg atgaggacaa caaccttctg 480 a 481 191 489 DNA Homo sapiensmisc_feature (1)...(489) n = A,T,C or G 191 atataaatta gactaagtgttttcaaataa atctaaatct tcagcatgat gtgttgtgta 60 taattggagt agatattaattaagtcccct gtataatgtt ttgtaatttt gcaaaacata 120 tcttgagttg tttaaacagtcaaaatgttt gatattttat accagcttat gagctcaaag 180 tactacagca aagcctagcctgcatatcat tcacccaaaa caaagtaata gcgcctcttt 240 tattattttg actgaatgttttatggaatt gaaagaaaca tacgttcttt tcaagacttc 300 ctcatgaatc tntcaattataggaaaagtt attgtgataa aataggaaca gctgaaagat 360 tgattaatga actattgttaattcttccta ttttaatgaa tgacattgaa ctgaattttt 420 tgtctgttaa atgaacttgatagctaataa aaagncaact agccatcaaa aaaaaaaaaa 480 aaaaaaaaa 489 192 516DNA Homo sapiens 192 acttcaaagc cagctgaagg aaagaggaag tgctagagagagcccccttc agtgtgcttc 60 tgacttttac ggacttggct tgttagaagg ctgaaagatgatggcaggaa tgaaaatcca 120 gcttgtatgc atgctactcc tggctttcag ctcctggagtctgtgctcag attcagaaga 180 ggaaatgaaa gcattagaag cagatttctt gaccaatatgcatacatcaa agattagtaa 240 agcacatgtt ccctcttgga agatgactct gctaaatgtttgcagtcttg taaataattt 300 gaacagccca gctgaggaaa caggagaagt tcatgaagaggagcttgttg caagaaggaa 360 cttcttactg ctttagatgg ctttagcttg gaagcaatgttgacaatata ccagctccac 420 aaaatctgtc acagcagggc ttttcaacac tgggagttaatccaggaaga tattcttgat 480 actggaaatg acaaaaatgg aaaggaagaa gtcata 516193 1409 DNA Homo sapiens 193 tgattctttt ccaaaacttt tagccatagggtcttttata gacagggata gtaaaatgaa 60 aattgagaaa tataagatga aaaggaatggtaaaaatatc ttttaggggg cttttaattg 120 gtgatctgaa atcttgggag aagctgttcttttcaggcct gaggtgctct tgactgtcgc 180 ctgcgcactg tgtaccccga gcaacattctaagggtgtgc tttcgccttg gctaactcct 240 ttgacctcat tcttcatata gtagtctaggaaaaagttgc aggtaattta aactgtctag 300 tggtacatag taactgaatt tctattcctatgagaaatga gaattattta tttgccatca 360 acacatttta tactttgcat ctccaaatttattgcggcga gacttgtcca ttgtgaaagt 420 tagagaacat tatgtttgta tcatttctttcataaaacct caagagcatt tttaagccct 480 tttcatcaga cccagtgaaa actaaggatagatgtttttt aactggaggt ctcctgataa 540 ggagaacaca atccaccatt gtcatttaagtaataagaca ggaaattgac cttgacgctt 600 tcttgttaaa tagatttaac aggaacatctgcacatcttt tttccttgtg cactatttgt 660 ttaattgcag tggattaata cagcaagagtgccacattat aactaggcaa ttatccattc 720 ttcaagactt agttattgtc acactaattgatcgtttaag gcataagatg gtctagcatt 780 aggaacatgt gaagctaatc tgctcaaaaagatcaacaaa ttaatattgt tgctgatatt 840 tgcataattg gctgcaatta tttaatgtttaattgggttg atcaaatgag attcagcaat 900 tcacaagtgc attaatataa acagaactggggcacttaaa atgataatga ttaacttata 960 ttgcatgttc tcttcctttc acttttttcagtgtctacat ttcagaccga gtttgtcagc 1020 ttttttgaaa acacatcagt agaaaccaagattttaaaat gaagtgtcaa gacgaaggca 1080 aaacctgagc agttcctaaa aagatttgctgttagaaatt ttctttgtgg cagtcattta 1140 ttaaggattc aactcgtgat acaccaaaagaagagttgac ttcagagatg tgttccatgc 1200 tctctagcac aggaatgaat aaatttataacacctgcttt agcctttgtt ttcaaaagca 1260 caaaggaaaa gtgaaaggga aagagaaacaagtgactgag aagtcttgtt aaggaatcag 1320 gttttttcta cctggtaaac attctctattcttttctcaa aagattgttg taagaaaaaa 1380 tgtaagmcaa aaaaaaaaaa aaaaaaaaa1409 194 441 DNA Homo sapiens 194 cagatttcgg tagccatctc cctccaaatatgtctctttc tgctttctta gtgcccatta 60 tttccccttc tcctttcttc tgtcactgccatctccttct tggtcttccc attgttcttt 120 aactggccgt aatgtggaat tgatatttacattttgatac ggtttttttc ttggcctgtg 180 tacgggattg cctcatttcc tgctctgaattttaaaatta gatattaaag ctgtcatatg 240 gtttcctcac aaaagtcaac aaagtccaaacaaaaatagt ttgccgtttt actttcatcc 300 attgaaaaag gaaattgtgc ctcttgcagcctaggcaaag gacatttagt actatcgatt 360 ctttccaccc tcacgatgac ttgcggttctctctgtagaa aagggatggc ctaagaaata 420 caactaaaaa aaaaaaaaaa a 441 195 707DNA Homo sapiens 195 cagaaaaata tttggaaaaa atataccact tcatagctaagtcttacaga gaagaggatt 60 tgctaataaa acttaagttt tgaaaattaa gatgcaggtagagcttctga actaatgccc 120 acagctccaa ggaagacatg tcctatttag ttattcaaatacaagttgag ggcattgtga 180 ttaagcaaac aatatatttg ttagaacttt gtttttaaattactgttcct tgacattact 240 tataaagagt ctctaacttt cgatttctaa aactatgtaatacaaaagta tagtttcccc 300 atttgataaa aggccaatga tactgagtag gatatatgcgtatcatgcta cttcattcag 360 tgtgtctgtt tttaatacta ataaggcagt ttgacagaaattatttcttt gggactaagg 420 tgattatcat ttttttcccc ttcaaaattg tgctttaagtgctgataacc acaggcagat 480 tgcaaagaac tgataaggca acaaaagtag agaattttaggatcaaaggc atgtaactga 540 aaggtaacaa cagtacataa gcgacaactg gggaaggcagcagtgaaaca tgtttgtggg 600 gttaagtgag tcattgtaaa taaggaattt gcacatttattttctgtcga cgcggccgcc 660 actgtgctgg atatctgcag aattccacca cactggactagtggatc 707 196 552 DNA Homo sapiens misc_feature (1)...(552) n = A,T,Cor G 196 tggccagcca gcctgatgtg gatggcttcc ttggggtggt gcttccctcaagcccgaatt 60 ngtggacatc atcaatgcca aacaatgagc cccatccatt ttccctacccttcctgccaa 120 gccagggant aagcagccca gaagcccagt aactgccctt tccctgcatatgcttttgat 180 ggtgtcatnt gctccttcct gtggcctcat ccaaactgta tnttcctttactgtttatat 240 nttcaccctg taatggttgg gaccaggcca atcccttntc cacttactataatggttgga 300 actaaacgtc accaaggtgg cttntccttg gctgaganat ggaaggcgtggtgggatttg 360 ctnctgggtt ccctaggccc tagtgagggc agaagagaaa ccatcctntcccttnttaca 420 ccgtgaggcc aagatcccct cagaaggcag gagtgctgcc ctntcccatggtgcccgtgc 480 ctntgtgctg tgtatgtgaa ccacccatgt gagggaataa acctggcactaggaaaaaaa 540 aaaaaaaaaa aa 552 197 449 DNA Homo sapiens misc_feature(1)...(449) n = A,T,C or G 197 ctccagagac aacttcgcgg tgtggtgaactctctgagga aaaacacgtg cgtggnanca 60 agtgactgag acctanaaat ccaagcgttggaggtcctga ggccagccta agtcgcttca 120 aaatggaacg aaggcgtttg cggggttccattcagagccg atacatcagc atgagtgtgt 180 ggacaagccc acggagactt gtggagctggcagggcagag cctgctgaag gatgaggccc 240 tggccattgc ccgccctgga gttgctgcccagggagctct tcccgccact cttcatggca 300 gcctttgacg ggagacacag ccagaccctgaaggcaatgg tgcaggcctg gcccttcacc 360 tgcctccctc tgggagtgct gatgaagggacaacatcttc acctggagac cttcaaagct 420 gtgcttgatg gacttgatgt gctccttgc 449198 606 DNA Homo sapiens 198 tgagtttgcc cccttacccc catcccagtg aatatttgcaattcctaaag acgtgttttg 60 attgtcacac ctgggtgggg aacatgctac tggcatctaatgcatagagg gcagtaatgc 120 tgctaaacat ctttcaacgc acaggacaga gccccacaaaagagaattat ctagccccaa 180 atgtccataa cactgctgtt gagaaaacct accgcaggatcttactgggc ttcataggta 240 agcttgcctt tgttctggct tctgtagata tataaaataaagacactgcc cagtccctcc 300 ctcaacgtcc cgagccaggg ctcaaggcaa ttccaataacagtagaatga acactaaata 360 ttgatttcaa aatctcagca actagaagaa tgaccaaccatcctggttgg cctgggactg 420 tcctagtttt agcattgaaa gtttcaggtt ccaggaaagccctcaggcct gggctgctgg 480 tcaccctagc agctgaggga ctcttcaata cagaattagtctttgtgcac tggagatgaa 540 tatactttaa tttgtaacat gtgaaaacat ctataaacatctactgaagc ctgttcttgt 600 ctgcac 606 199 369 DNA Homo sapiensmisc_feature (1)...(369) n = A,T,C or G 199 ggcaactttt tgcggattgttcttgcttnc aggctttgcg ctgcaaatcc agtgctacca 60 gtgtgaagaa ttccagctgaacaacgactg ctcctccccc gagttcattg tgaattgcac 120 ggtgaacgtt caagacatgtgtcagaaaga agtgatggag caaagtgccg ggatcatgta 180 ccgcaagtcc tgtgcatcatcagcggcctg tctcatcgcc tctgccgggt accagtcctt 240 ctgctcccca gggaaactgaactcagtttg catcagctgc tgcaacaccc ctctttgtaa 300 cgggccaagg cccaagaaaaggggaagttc tgcctcggcc ctcangccat ggctccgcac 360 caccatcct 369 200 55 PRTHomo sapiens 200 Met Tyr Arg Asn Trp Ser Gly Cys Phe Gly Leu Gln Val ThrLeu Cys 5 10 15 His Thr Phe Glu Thr Arg Asp Leu Ser Arg Leu Ser Ser AspSer Gln 20 25 30 Pro Thr Ser Asn Val Ser Gln Ser Ile Ser His Lys Val LeuSer Phe 35 40 45 Ser Gly Val Ile Val Thr Pro 50 55 201 67 PRT Homosapiens 201 Met Gln Leu Leu Ser Pro Asn Thr Lys Phe Thr Ser Cys Leu SerArg 5 10 15 Gln Arg Gly Asn Leu Val Phe Leu Gly Asp Leu Lys Gly Cys SerGlu 20 25 30 Leu Lys Asn Phe Gln Glu Leu Ile Asn Gln Ser Ala Leu Val HisPro 35 40 45 Arg Val Asp Val Trp Trp Tyr Cys Gly Gly Pro Leu Leu Gly ThrLeu 50 55 60 Pro Asn Asn 65 202 73 PRT Homo sapiens 202 Met Thr Pro GluLys Leu Arg Thr Leu Cys Glu Ile Asp Trp Leu Thr 5 10 15 Leu Glu Val GlyTrp Leu Ser Glu Glu Ser Leu Glu Arg Ser Leu Val 20 25 30 Ser Lys Val TrpHis Lys Val Thr Cys Lys Pro Lys His Pro Asp Gln 35 40 45 Phe Leu Tyr IleAsp Ser Tyr Ser Trp Phe Arg Pro Leu Pro Pro Leu 50 55 60 Pro Thr Val ValLys Arg Thr Ala Ala 65 70 203 2008 DNA Homo sapiens 203 ctccagagacaacttcgcgg tgtggtgaac tctctgagga aaaacacgtg cgtggtaaca 60 agtgactgagacctagaaat ccaagcgttg gaggtcctga ggccagccta agtcgcttca 120 aaatggaacgaaggcgtttg cggggttcca ttcagagccg atacatcagc atgagtgtgt 180 ggacaagcccacggagactt gtggagctgg cagggcagag cctgctgaag gatgaggccc 240 tggccattgcccgccctgga gttgctgccc agggagctct tcccgccact cttcatggca 300 gcctttgacgggagacacag ccagaccctg aaggcaatgg tgcaggcctg gcccttcacc 360 tgcctccctctgggagtgct gatgaaggga caacatcttc acctggagac cttcaaagct 420 gtgcttgatggacttgatgt gctccttgcc caggaggttc gccccaggag gtggaaactt 480 caagtgctggatttacggaa gaactctcat caggacttct ggactgtatg gtctggaaac 540 agggccagtctgtactcatt tccagagcca gaagcagctc agcccatgac aaagaagcga 600 aaagtagatggtttgagcac agaggcagag cagcccttca ttccagtaga ggtgctcgta 660 gacctgttcctcaaggaagg tgcctgtgat gaattgttct cctacctcat tgagaaagtg 720 aagcgaaagaaaaatgtact acgcctgtgc tgtaagaagc tgaagatttt tgcaatgccc 780 atgcaggatatcaagatgat cctgaaaatg gtgcagctgg actctattga agatttggaa 840 gtgacttgtacctggaagct acccaccttg gcgaaatttt ctccttacct gggccagatg 900 attaatctgcgtagactcct cctctcccac atccatgcat cttcctacat ttccccggag 960 aaggaagagcagtatatcgc ccagttcacc tctcagttcc tcagtctgca gtgcctgcag 1020 gctctctatgtggactcttt atttttcctt agaggccgcc tggatcagtt gctcaggcac 1080 gtgatgaaccccttggaaac cctctcaata actaactgcc ggctttcgga aggggatgtg 1140 atgcatctgtcccagagtcc cagcgtcagt cagctaagtg tcctgagtct aagtggggtc 1200 atgctgaccgatgtaagtcc cgagcccctc caagctctgc tggagagagc ctctgccacc 1260 ctccaggacctggtctttga tgagtgtggg atcacggatg atcagctcct tgccctcctg 1320 ccttccctgagccactgctc ccagcttaca accttaagct tctacgggaa ttccatctcc 1380 atatctgccttgcagagtct cctgcagcac ctcatcgggc tgagcaatct gacccacgtg 1440 ctgtatcctgtccccctgga gagttatgag gacatccatg gtaccctcca cctggagagg 1500 cttgcctatctgcatgccag gctcagggag ttgctgtgtg agttggggcg gcccagcatg 1560 gtctggcttagtgccaaccc ctgtcctcac tgtggggaca gaaccttcta tgacccggag 1620 cccatcctgtgcccctgttt catgcctaac tagctgggtg cacatatcaa atgcttcatt 1680 ctgcatacttggacactaaa gccaggatgt gcatgcatct tgaagcaaca aagcagccac 1740 agtttcagacaaatgttcag tgtgagtgag gaaaacatgt tcagtgagga aaaaacattc 1800 agacaaatgttcagtgagga aaaaaagggg aagttgggga taggcagatg ttgacttgag 1860 gagttaatgtgatctttggg gagatacatc ttatagagtt agaaatagaa tctgaatttc 1920 taaagggagattctggcttg ggaagtacat gtaggagtta atccctgtgt agactgttgt 1980 aaagaaactgttgaaaaaaa aaaaaaaa 2008 204 923 DNA Homo sapiens 204 tgagtttgcccccttacccc catcccagtg aatatttgca attcctaaag acgtgttttg 60 attgtcacacctgggtgggg aacatgctac tggcatctaa tgcatagagg gcagtaatgc 120 tgctaaacatctttcaacgc acaggacaga gccccacaaa agagaattat ctagccccaa 180 atgtccataacactgctgtt gagaaaacct accgcaggat cttactgggc ttcataggta 240 agcttgcctttgttctggct tctgtagata tataaaataa agacactgcc cagtccctcc 300 ctcaacgtcccgagccaggg ctcaaggcaa ttccaataac agtagaatga acactaaata 360 ttgatttcaaaatctcagca actagaagaa tgaccaacca tcctggttgg cctgggactg 420 tcctagttttagcattgaaa gtttcaggtt ccaggaaagc cctcaggcct gggctgctgg 480 tcaccctagcagctgaggga ctcttcaata cagaattagt ctttgtgcac tggagatgaa 540 tatactttaatttgtaacat gtgaaaacat ctataaacat ctactgaagc ctgttctgtc 600 tgcaccgacattttcattga gtacggattc ttcctaccag atacagctgc tctacaactt 660 tcgagggctggtataaaact agcttttacc tatttttaaa aattacatga atagtaaaaa 720 cttggattaacccagtattc gggtattttc aatttccttg ggagcttaga ggacggacaa 780 ataaaaagattatttcaaca tcaaatatat gctattgttt acatatgaag ataaccacat 840 atatgtataaattcaccgtt actttttagc aatactataa aatccaacag aaaaaaatag 900 catttactaaaaaaaaaaaa aaa 923 205 1619 DNA Homo sapiens 205 ggcaactttt tgcggattgttcttgcttcc aggctttgcg ctgcaaatcc agtgctacca 60 gtgtgaagaa ttccagctgaacaacgactg ctcctccccc gagttcattg tgaattgcac 120 ggtgaacgtt caagacatgtgtcagaaaga agtgatggag caaagtgccg ggatcatgta 180 ccgcaagtcc tgtgcatcatcagcggcctg tctcatcgcc tctgccgggt accagtcctt 240 ctgctcccca gggaaactgaactcagtttg catcagctgc tgcaacaccc ctctttgtaa 300 cgggccaagg cccaagaaaaggggaagttc tgcctcggcc ctcaggccag ggctccgcac 360 caccatcctg ttcctcaaattagccctctt ctcggcacac tgctgaagct gaaggagatg 420 ccaccccctc ctgcattgttcttccagccc tcgcccccaa ccccccacct ccctgagtga 480 gtttcttctg ggtgtccttttattctgggt agggagcggg agtccgtgtt ctcttttgtt 540 cctgtgcaaa taatgaaagagctcggtaaa gcattctgaa taaattcagc ctgactgaat 600 tttcagtatg tacttgaaggaaggaggtgg agtgaaagtt cacccccatg tctgtgtaac 660 cggagtcaag gccaggctggcagagtcagt ccttagaagt cactgaggtg ggcatctgcc 720 ttttgtaaag cctccagtgtccattccatc cctgatgggg gcatagtttg agactgcaga 780 gtgagagtga cgttttcttagggctggagg gccagttccc actcaaggct ccctcgcttg 840 acattcaaac ttcatgctcctgaaaaccat tctctgcagc agaattggct ggtttcgcgc 900 ctgagttggg ctctagtgactcgagactca atgactggga cttagactgg ggctcggcct 960 cgctctgaaa agtgcttaagaaaatcttct cagttctcct tgcagaggac tggcgccggg 1020 acgcgaagag caacgggcgctgcacaaagc gggcgctgtc ggtggtggag tgcgcatgta 1080 cgcgcaggcg cttctcgtggttggcgtgct gcagcgacag gcggcagcac agcaccttgc 1140 acgaacaccc gccgaaactgctgcgaggac accgtgtaca ggagcgggtt gatgaccgag 1200 ctgaggtaga aaaacgtctccgagaagggg aggaggatca tgtacgcccg gaagtaggac 1260 ctcgtccagt cgtgcttgggtttggccgca gccatgatcc tccgaatctg gttgggcatc 1320 cagcatacgg ccaatgtcacaacaatcagc cctgggcaga cacgagcagg agggagagac 1380 agagaaaaga aaaacacagcatgagaacac agtaaatgaa taaaaccata aaatatttag 1440 cccctctgtt ctgtgcttactggccaggaa atggtaccaa tttttcagtg ttggacttga 1500 cagcttcttt tgccacaagcaagagagaat ttaacactgt ttcaaacccg ggggagttgg 1560 ctgtgttaaa gaaagaccattaaatgcttt agacagtgta aaaaaaaaaa aaaaaaaaa 1619 206 2364 DNA Homosapiens 206 atgcagcatc accaccatca ccacttctcc gacgagaccc tggacaaagtgcccaagtca 60 gagggctact gtagccgtat cctgcgcgcc cagggcacgc ggcgcgagggctacaccgag 120 ttcagcctcc gcgtggaggg cgaccccgac ttctacaagc cgggaaccagctaccgcgta 180 acactttcag ctgctcctcc ctcctacttc agaggattca cattaattgccctcagagag 240 aacagagagg gtgataagga agaagaccat gctgggacct tccagatcatagacgaagaa 300 gaaactcagt ttatgagcaa ttgccctgtt gcagtcactg aaagcactccacggaggagg 360 acccggatcc aggtgttttg gatagcacca ccagcgggaa caggctgcgtgattctgaag 420 gccagcatcg tacaaaaacg cattatttat tttcaagatg agggctctctgaccaagaaa 480 ctttgtgaac aagattccac atttgatggg gtgactgaca aacccatcttagactgctgt 540 gcctgcggaa ctgccaagta cagactcaca ttttatggga attggtccgagaagacacac 600 ccaaaggatt accctcgtcg ggccaaccac tggtctgcga tcatcggaggatcccactcc 660 aagaattatg tactgtggga atatggagga tatgccagcg aaggcgtcaaacaagttgca 720 gaattgggct cacccgtgaa aatggaggaa gaaattcgac aacagagtgatgaggtcctc 780 accgtcatca aagccaaagc ccagtggcca gcctggcagc ctctcaacgtgagagcagca 840 ccttcagctg aattttccgt ggacagaacg cgccatttaa tgtccttcctgaccatgatg 900 ggccctagtc ccgactggaa cgtaggctta tctgcagaag atctgtgcaccaaggaatgt 960 ggctgggtcc agaaggtggt gcaagacctg attccctggg acgctggcaccgacagcggg 1020 gtgacctatg agtcacccaa caaacccacc attccccagg agaaaatccggcccctgacc 1080 agcctggacc atcctcagag tcctttctat gacccagagg gtgggtccatcactcaagta 1140 gccagagttg tcatcgagag aatcgcacgg aagggtgaac aatgcaatattgtacctgac 1200 aatgtcgatg atattgtagc tgacctggct ccagaagaga aagatgaagatgacacccct 1260 gaaacctgca tctactccaa ctggtcccca tggtccgcct gcagctcctccacctgtgac 1320 aaaggcaaga ggatgcgaca gcgcatgctg aaagcacagc tggacctcagcgtcccctgc 1380 cctgacaccc aggacttcca gccctgcatg ggccctggct gcagtgacgaagacggctcc 1440 acctgcacca tgtccgagtg gatcacctgg tcgccctgca gcatctcctgcggcatgggc 1500 atgaggtccc gggagaggta tgtgaagcag ttcccggagg acggctccgtgtgcacgctg 1560 cccactgagg aaacggagaa gtgcacggtc aacgaggagt gctctcccagcagctgcctg 1620 atgaccgagt ggggcgagtg ggacgagtgc agcgccacct gcggcatgggcatgaagaag 1680 cggcaccgca tgatcaagat gaaccccgca gatggctcca tgtgcaaagccgagacatca 1740 caggcagaga agtgcatgat gccagagtgc cacaccatcc catgcttgctgtccccatgg 1800 tccgagtgga gtgactgcag cgtgacctgc gggaagggca tgcgaacccgacagcggatg 1860 ctcaagtctc tggcagaact tggagactgc aatgaggatc tggagcaggtggagaagtgc 1920 atgctccctg aatgccccat tgactgtgag ctcaccgagt ggtcccagtggtcggaatgt 1980 aacaagtcat gtgggaaagg ccacgtgatt cgaacccgga tgatccaaatggagcctcag 2040 tttggaggtg caccctgccc agagactgtg cagcgaaaaa agtgccgcatccgaaaatgc 2100 cttcgaaatc catccatcca aaagctacgc tggagggagg cccgagagagccggcggagt 2160 gagcagctga aggaagagtc tgaaggggag cagttcccag gttgtaggatgcgcccatgg 2220 acggcctggt cagaatgcac caaactgtgc ggaggtggaa ttcaggaacgttacatgact 2280 gtaaagaaga gattcaaaag ctcccagttt accagctgca aagacaagaaggagatcaga 2340 gcatgcaatg ttcatccttg ttag 2364 207 787 PRT Homo sapiens207 Met Gln His His His His His His Phe Ser Asp Glu Thr Leu Asp Lys 5 1015 Val Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu Arg Ala Gln Gly 20 2530 Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg Val Glu Gly Asp 35 4045 Pro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val Thr Leu Ser Ala 50 5560 Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile Ala Leu Arg Glu 65 7075 80 Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly Thr Phe Gln Ile 8590 95 Ile Asp Glu Glu Glu Thr Gln Phe Met Ser Asn Cys Pro Val Ala Val100 105 110 Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg Ile Gln Val Phe TrpIle 115 120 125 Ala Pro Pro Ala Gly Thr Gly Cys Val Ile Leu Lys Ala SerIle Val 130 135 140 Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser LeuThr Lys Lys 145 150 155 160 Leu Cys Glu Gln Asp Ser Thr Phe Asp Gly ValThr Asp Lys Pro Ile 165 170 175 Leu Asp Cys Cys Ala Cys Gly Thr Ala LysTyr Arg Leu Thr Phe Tyr 180 185 190 Gly Asn Trp Ser Glu Lys Thr His ProLys Asp Tyr Pro Arg Arg Ala 195 200 205 Asn His Trp Ser Ala Ile Ile GlyGly Ser His Ser Lys Asn Tyr Val 210 215 220 Leu Trp Glu Tyr Gly Gly TyrAla Ser Glu Gly Val Lys Gln Val Ala 225 230 235 240 Glu Leu Gly Ser ProVal Lys Met Glu Glu Glu Ile Arg Gln Gln Ser 245 250 255 Asp Glu Val LeuThr Val Ile Lys Ala Lys Ala Gln Trp Pro Ala Trp 260 265 270 Gln Pro LeuAsn Val Arg Ala Ala Pro Ser Ala Glu Phe Ser Val Asp 275 280 285 Arg ThrArg His Leu Met Ser Phe Leu Thr Met Met Gly Pro Ser Pro 290 295 300 AspTrp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys Thr Lys Glu Cys 305 310 315320 Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro Trp Asp Ala Gly 325330 335 Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys Pro Thr Ile Pro340 345 350 Gln Glu Lys Ile Arg Pro Leu Thr Ser Leu Asp His Pro Gln SerPro 355 360 365 Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gln Val Ala ArgVal Val 370 375 380 Ile Glu Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn IleVal Pro Asp 385 390 395 400 Asn Val Asp Asp Ile Val Ala Asp Leu Ala ProGlu Glu Lys Asp Glu 405 410 415 Asp Asp Thr Pro Glu Thr Cys Ile Tyr SerAsn Trp Ser Pro Trp Ser 420 425 430 Ala Cys Ser Ser Ser Thr Cys Asp LysGly Lys Arg Met Arg Gln Arg 435 440 445 Met Leu Lys Ala Gln Leu Asp LeuSer Val Pro Cys Pro Asp Thr Gln 450 455 460 Asp Phe Gln Pro Cys Met GlyPro Gly Cys Ser Asp Glu Asp Gly Ser 465 470 475 480 Thr Cys Thr Met SerGlu Trp Ile Thr Trp Ser Pro Cys Ser Ile Ser 485 490 495 Cys Gly Met GlyMet Arg Ser Arg Glu Arg Tyr Val Lys Gln Phe Pro 500 505 510 Glu Asp GlySer Val Cys Thr Leu Pro Thr Glu Glu Thr Glu Lys Cys 515 520 525 Thr ValAsn Glu Glu Cys Ser Pro Ser Ser Cys Leu Met Thr Glu Trp 530 535 540 GlyGlu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met Gly Met Lys Lys 545 550 555560 Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly Ser Met Cys Lys 565570 575 Ala Glu Thr Ser Gln Ala Glu Lys Cys Met Met Pro Glu Cys His Thr580 585 590 Ile Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser Asp Cys SerVal 595 600 605 Thr Cys Gly Lys Gly Met Arg Thr Arg Gln Arg Met Leu LysSer Leu 610 615 620 Ala Glu Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln ValGlu Lys Cys 625 630 635 640 Met Leu Pro Glu Cys Pro Ile Asp Cys Glu LeuThr Glu Trp Ser Gln 645 650 655 Trp Ser Glu Cys Asn Lys Ser Cys Gly LysGly His Val Ile Arg Thr 660 665 670 Arg Met Ile Gln Met Glu Pro Gln PheGly Gly Ala Pro Cys Pro Glu 675 680 685 Thr Val Gln Arg Lys Lys Cys ArgIle Arg Lys Cys Leu Arg Asn Pro 690 695 700 Ser Ile Gln Lys Leu Arg TrpArg Glu Ala Arg Glu Ser Arg Arg Ser 705 710 715 720 Glu Gln Leu Lys GluGlu Ser Glu Gly Glu Gln Phe Pro Gly Cys Arg 725 730 735 Met Arg Pro TrpThr Ala Trp Ser Glu Cys Thr Lys Leu Cys Gly Gly 740 745 750 Gly Ile GlnGlu Arg Tyr Met Thr Val Lys Lys Arg Phe Lys Ser Ser 755 760 765 Gln PheThr Ser Cys Lys Asp Lys Lys Glu Ile Arg Ala Cys Asn Val 770 775 780 HisPro Cys 785 208 1362 DNA Homo sapiens 208 atggcttcac ccagcctcccgggcagtgac tgctcccaaa tcattgatca cagtcatgtc 60 cccgagtttg aggtggccacctggatcaaa atcaccctta ttctggtgta cctgatcatc 120 ttcgtgatgg gccttctggggaacagcgcc accattcggg tcacccaggt gctgcagaag 180 aaaggatact tgcagaaggaggtgacagac cacatggtga gtttggcttg ctcggacatc 240 ttggtgttcc tcatcggcatgcccatggag ttctacagca tcatctggaa tcccctgacc 300 acgtccagct acaccctgtcctgcaagctg cacactttcc tcttcgaggc ctgcagctac 360 gctacgctgc tgcacgtgctgacactcagc tttgagcgct acatcgccat ctgtcacccc 420 ttcaggtaca aggctgtgtcgggaccttgc caggtgaagc tgctgattgg cttcgtctgg 480 gtcacctccg ccctggtggcactgcccttg ctgtttgcca tgggtactga gtaccccctg 540 gtgaacgtgc ccagccaccggggtctcact tgcaaccgct ccagcacccg ccaccacgag 600 cagcccgaga cctccaatatgtccatctgt accaacctct ccagccgctg gaccgtgttc 660 cagtccagca tcttcggcgccttcgtggtc tacctcgtgg tcctgctctc cgtagccttc 720 atgtgctgga acatgatgcaggtgctcatg aaaagccaga agggctcgct ggccgggggc 780 acgcggcctc cgcagctgaggaagtccgag agcgaagaga gcaggaccgc caggaggcag 840 accatcatct tcctgaggctgattgttgtg acattggccg tatgctggat gcccaaccag 900 attcggagga tcatggctgcggccaaaccc aagcacgact ggacgaggtc ctacttccgg 960 gcgtacatga tcctcctccccttctcggag acgtttttct acctcagctc ggtcatcaac 1020 ccgctcctgt acacggtgtcctcgcagcag tttcggcggg tgttcgtgca ggtgctgtgc 1080 tgccgcctgt cgctgcagcacgccaaccac gagaagcgcc tgcgcgtaca tgcgcactcc 1140 accaccgaca gcgcccgctttgtgcagcgc ccgttgctct tcgcgtcccg gcgccagtcc 1200 tctgcaagga gaactgagaagattttctta agcacttttc agagcgaggc cgagccccag 1260 tctaagtccc agtcattgagtctcgagtca ctagagccca actcaggcgc gaaaccagcc 1320 aattctgctg cagagaatggttttcaggag catgaagttt ga 1362 209 453 PRT Homo sapiens 209 Met Ala SerPro Ser Leu Pro Gly Ser Asp Cys Ser Gln Ile Ile Asp 5 10 15 His Ser HisVal Pro Glu Phe Glu Val Ala Thr Trp Ile Lys Ile Thr 20 25 30 Leu Ile LeuVal Tyr Leu Ile Ile Phe Val Met Gly Leu Leu Gly Asn 35 40 45 Ser Ala ThrIle Arg Val Thr Gln Val Leu Gln Lys Lys Gly Tyr Leu 50 55 60 Gln Lys GluVal Thr Asp His Met Val Ser Leu Ala Cys Ser Asp Ile 65 70 75 80 Leu ValPhe Leu Ile Gly Met Pro Met Glu Phe Tyr Ser Ile Ile Trp 85 90 95 Asn ProLeu Thr Thr Ser Ser Tyr Thr Leu Ser Cys Lys Leu His Thr 100 105 110 PheLeu Phe Glu Ala Cys Ser Tyr Ala Thr Leu Leu His Val Leu Thr 115 120 125Leu Ser Phe Glu Arg Tyr Ile Ala Ile Cys His Pro Phe Arg Tyr Lys 130 135140 Ala Val Ser Gly Pro Cys Gln Val Lys Leu Leu Ile Gly Phe Val Trp 145150 155 160 Val Thr Ser Ala Leu Val Ala Leu Pro Leu Leu Phe Ala Met GlyThr 165 170 175 Glu Tyr Pro Leu Val Asn Val Pro Ser His Arg Gly Leu ThrCys Asn 180 185 190 Arg Ser Ser Thr Arg His His Glu Gln Pro Glu Thr SerAsn Met Ser 195 200 205 Ile Cys Thr Asn Leu Ser Ser Arg Trp Thr Val PheGln Ser Ser Ile 210 215 220 Phe Gly Ala Phe Val Val Tyr Leu Val Val LeuLeu Ser Val Ala Phe 225 230 235 240 Met Cys Trp Asn Met Met Gln Val LeuMet Lys Ser Gln Lys Gly Ser 245 250 255 Leu Ala Gly Gly Thr Arg Pro ProGln Leu Arg Lys Ser Glu Ser Glu 260 265 270 Glu Ser Arg Thr Ala Arg ArgGln Thr Ile Ile Phe Leu Arg Leu Ile 275 280 285 Val Val Thr Leu Ala ValCys Trp Met Pro Asn Gln Ile Arg Arg Ile 290 295 300 Met Ala Ala Ala LysPro Lys His Asp Trp Thr Arg Ser Tyr Phe Arg 305 310 315 320 Ala Tyr MetIle Leu Leu Pro Phe Ser Glu Thr Phe Phe Tyr Leu Ser 325 330 335 Ser ValIle Asn Pro Leu Leu Tyr Thr Val Ser Ser Gln Gln Phe Arg 340 345 350 ArgVal Phe Val Gln Val Leu Cys Cys Arg Leu Ser Leu Gln His Ala 355 360 365Asn His Glu Lys Arg Leu Arg Val His Ala His Ser Thr Thr Asp Ser 370 375380 Ala Arg Phe Val Gln Arg Pro Leu Leu Phe Ala Ser Arg Arg Gln Ser 385390 395 400 Ser Ala Arg Arg Thr Glu Lys Ile Phe Leu Ser Thr Phe Gln SerGlu 405 410 415 Ala Glu Pro Gln Ser Lys Ser Gln Ser Leu Ser Leu Glu SerLeu Glu 420 425 430 Pro Asn Ser Gly Ala Lys Pro Ala Asn Ser Ala Ala GluAsn Gly Phe 435 440 445 Gln Glu His Glu Val 450 210 625 DNA Homo sapiensmisc_feature (1)...(625) n = A,T,C or G 210 agttctcctt gcagaggactggcgccggga cgcgaagagc aacgggcgct gcacaaagcg 60 ggcgctgtcg gtggtggagtgcgcatgtac gcgcaggcgc ttctcgtggt tggcgtgctg 120 cagcgacagg cggcagcacagcacctgcac gaacacccgc cgaaactgct gcgaggacac 180 cgtgtacagg agcgggttgatgaccgagct gaggtagaaa aacgtctccg agaaggggag 240 gaggatcatg tacgcccggaagtaggacct cgtccagtcg tgcttgggtt tggccgcagc 300 catgatcctc cgaatctggttgggcatcca gcatacggcc aatgtcacaa caatcagccc 360 tgggcagaca cgagcaggagggagagacag agaaaagaaa aacacagcat gagaacacag 420 taaatgaata aaaccataaaatatttagcc cctctgttct gtgcttactg gccaggaaat 480 ggtaccaatt tttcagtgttggacttgaca gcttcttttg ccacaagcaa gagagaattt 540 aacactgttt caaacccgggggagttggct gtgttaaaga aagaccatta aatgctttag 600 acagtgnaaa aaaaaaaaaaaaaaa 625 211 1619 DNA Homo sapiens 211 ggcaactttt tgcggattgt tcttgcttccaggctttgcg ctgcaaatcc agtgctacca 60 gtgtgaagaa ttccagctga acaacgactgctcctccccc gagttcattg tgaattgcac 120 ggtgaacgtt caagacatgt gtcagaaagaagtgatggag caaagtgccg ggatcatgta 180 ccgcaagtcc tgtgcatcat cagcggcctgtctcatcgcc tctgccgggt accagtcctt 240 ctgctcccca gggaaactga actcagtttgcatcagctgc tgcaacaccc ctctttgtaa 300 cgggccaagg cccaagaaaa ggggaagttctgcctcggcc ctcaggccag ggctccgcac 360 caccatcctg ttcctcaaat tagccctcttctcggcacac tgctgaagct gaaggagatg 420 ccaccccctc ctgcattgtt cttccagccctcgcccccaa ccccccacct ccctgagtga 480 gtttcttctg ggtgtccttt tattctgggtagggagcggg agtccgtgtt ctcttttgtt 540 cctgtgcaaa taatgaaaga gctcggtaaagcattctgaa taaattcagc ctgactgaat 600 tttcagtatg tacttgaagg aaggaggtggagtgaaagtt cacccccatg tctgtgtaac 660 cggagtcaag gccaggctgg cagagtcagtccttagaagt cactgaggtg ggcatctgcc 720 ttttgtaaag cctccagtgt ccattccatccctgatgggg gcatagtttg agactgcaga 780 gtgagagtga cgttttctta gggctggagggccagttccc actcaaggct ccctcgcttg 840 acattcaaac ttcatgctcc tgaaaaccattctctgcagc agaattggct ggtttcgcgc 900 ctgagttggg ctctagtgac tcgagactcaatgactggga cttagactgg ggctcggcct 960 cgctctgaaa agtgcttaag aaaatcttctcagttctcct tgcagaggac tggcgccggg 1020 acgcgaagag caacgggcgc tgcacaaagcgggcgctgtc ggtggtggag tgcgcatgta 1080 cgcgcaggcg cttctcgtgg ttggcgtgctgcagcgacag gcggcagcac agcaccttgc 1140 acgaacaccc gccgaaactg ctgcgaggacaccgtgtaca ggagcgggtt gatgaccgag 1200 ctgaggtaga aaaacgtctc cgagaaggggaggaggatca tgtacgcccg gaagtaggac 1260 ctcgtccagt cgtgcttggg tttggccgcagccatgatcc tccgaatctg gttgggcatc 1320 cagcatacgg ccaatgtcac aacaatcagccctgggcaga cacgagcagg agggagagac 1380 agagaaaaga aaaacacagc atgagaacacagtaaatgaa taaaaccata aaatatttag 1440 cccctctgtt ctgtgcttac tggccaggaaatggtaccaa tttttcagtg ttggacttga 1500 cagcttcttt tgccacaagc aagagagaatttaacactgt ttcaaacccg ggggagttgg 1560 ctgtgttaaa gaaagaccat taaatgctttagacagtgta aaaaaaaaaa aaaaaaaaa 1619 212 1010 DNA Homo sapiens 212ccgcagccgg gagcccgagc gcgggcgatg caggctccgc gagcggcacc tgcggctcct 60ctaagctacg accgtcgtct ccgctggcag cagctgcggg ccccagcagc ctcggcagcc 120acagccgctg cagcctgggg cagcctccgc tgctgtcgcc tcctctgatg cgcttgccct 180ctccctggcc ccgggactcc gggagaatgt gggtcctagg catcgcggca actttttgcg 240gattgttctt gcttccaagg ctttgcgctg caaatccagt gctaccagtg tgaagaattc 300cagctgaaca acgactgctc ctcccccgag ttcattgtga attgcacggt gaacgttcaa 360gacatgtgtc agaaagaagt gatggagcaa agtgccggga tcatgtaccg caagtcctgt 420gcatcatcag cggcctgtct catcgcctct gccgggtacc agtccttctg ctccccaggg 480aaactgaact cagtttgcat cagctgctgc aacacccctc tttgtaaccg ggccaaggcc 540caagaaaagg ggaagttctg cctcggccct caggccaggg ctccgaacca ccatcctgtc 600cctcaaatta agccctactt ctcggcacac tgctggaagc ttgaagggag aaggcaccca 660ctcctgcata gtccatccag gcctcgcccc acacacccca ctccctgaga gagcacgccc 720agggagacca aaaaccggga taggcaacgg acccccagac accacaaggg acccgaggac 780aaagacgcag acaactcgcg aaagccaccc acgaatacaa cggcccgaac acagatataa 840cgcacgagcc ccgaccgaca agagaagaag cagaagaaac acccacagac agaaacagac 900accagcaaca agcgaaaaca gcaaaacgac actagcgaga caccacctgc acacaacacc 960acagcccaac acagaggaca cgacaacaaa gagacagcac caacgacgaa 1010 213 480 DNAHomo sapiens 213 gccaactccg gaggctctgg tgctcggccc gggagcgcga gcgggaggagcagagacccg 60 cagccgggag cccgagcgcg ggcgatgcag gctccgcgag cggcacctgcggctcctcta 120 agctacgacc gtcgtctccg cggcagcagc gcgggcccca gcagcctcggcagccacagc 180 cgctgcagcc ggggcagcct ccgctgctgt cgcctcctct gatgcgcttgccctctcccg 240 gccccgggac tccgggagaa tgtgggtcct aggcatcgcg gcaactttttgcggattgtt 300 cttgcttcca ggctttgcgc tgcaaatcca gtgctaccag tgtgaagaattccagctgaa 360 caacgactgc tcctcccccg agttcattgt gaattgcacg gtgaacgttcaagacatgtg 420 tgagaaagaa gtgatggagc aaagtgccgg gatcatgtac cgcaagtcctgtgcatgatc 480 214 1897 DNA Homo sapiens misc_feature (1)...(1897) n =A,T,C or G 214 gccaactccg gaggctctgg tgctcggccc gggagcgcga gcgggaggagcagagacccg 60 cagccgggag cccgagcgcg ggcgatgcag gctccgcgag cggcacctgcggctcctcta 120 agctacgacc gtcgtctccg cggcagcagc gcgggcccca gcagcctcggcagccacagc 180 cgctgcagcc ggggcagcct ccgctgctgt cgcctcctct gatgcgcttgccctctcccg 240 gccccgggac tccgggagaa tgtgggtcct aggcatcgcg gcaactttttgcggattgtt 300 cttgcttcca ggctttgcgc tgcaaatcca gtgctaccag tgtgaagaattccagctgaa 360 caacgactgc tcctcccccg agttcattgt gaattgcacg gtgaacgttcaagacatgtg 420 tcagaaagaa gtgatggagc aaagtgccgg gatcatgtac cgcaagtcctgtgcatcatc 480 agcggcctgt ctcatcgcct ctgccgggta ccagtccttc tgctccccagggaaactgaa 540 ctcagtttgc atcagctgct gcaacacccc tctttgtaac gggccaaggcccaagaaaag 600 gggaagttct gcctcggccc tcaggccagg gctccgcacc accatcctgttcctcaaatt 660 agccctcttc tcggcacact gctgaagctg aaggagatgc caccccctcctgcattgttc 720 ttccagccct cgcccccaac cccccacctc cctgagtgag tttcttctgggtgtcctttt 780 attctgggta gggagcggga gtccgtgttc tcttttgttc ctgtgcaaataatgaaagag 840 ctcggtaaag cattctgaat aaattcagcy tgactgaatt ttcagtatgtacttgaagga 900 aggaggtgga gtgaaagttc acccccatgt ctgtgtaacc ggagtcaaggccaggctggc 960 agagtcwgtc cttagaagtc actgaggtgg gcatctgcct tttgtaaagcctccagtgtc 1020 cattccatcc ctgatggggg catagtttga gactgcagag tgagagtgacgttttcttag 1080 ggctggaggg ccagttccca ctcaaggctc cctcgcttga cattcaaacttcatgctcct 1140 gaaaaccatt ctctgcagca gaattggctg gtttcgcgcc tgagttgggctctagtgact 1200 cgagactcaa tgactgggac ttagactggg gctcggcctc gctctgaaaagtgcttaaga 1260 aaatcttctc agttctcctt gcagaggact ggcgccggga cgcgaagagcaacgggcgct 1320 gcacaaagcg ggcgctgtcg gtggtggagt gcgcatgtac gcgcaggcgcttctcgtggt 1380 tggcgtgctg cagcgacagg cggcagcaca gcacctgcac gaacacccgccgaaactgct 1440 gcgaggacac cgtgtacagg agcgggttga tgaccgagct gaggtagaaaaacgtctccg 1500 agaaggggag gaggatcatg tacgcccgga agtaggacct cgtccagtcgtgcttgggtt 1560 tggccgcagc catgatcctc cgaatctggt tgggcatcca gcatacggccaatgtcacaa 1620 caatcagccc tgggcagaca cgagcaggag ggagagacag agaaaagaaaaacacagcat 1680 gagaacacag taaatgaata aaaccataaa atatttagcc cctctgttctgtgcttactg 1740 gccaggaaat ggtaccaatt tttcagtgtt ggacttgaca gcttcttttgccacaagcaa 1800 gagagaattt aacactgttt caaacccggg ggagttggct gtgttaaagaaagaccatta 1860 aatgctttag acagtgtaaa aaaaaaaaaa aaaaaaa 1897 215 141PRT Homo sapiens 215 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly LeuPhe Leu Leu 5 10 15 Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys GluGlu Phe Gln 20 25 30 Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val AsnCys Thr Val 35 40 45 Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu GlnSer Ala Gly 50 55 60 Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala Ala CysLeu Ile Ala 65 70 75 80 Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly LysLeu Asn Ser Val 85 90 95 Cys Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn GlyPro Arg Pro Lys 100 105 110 Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg ProGly Leu Arg Thr Thr 115 120 125 Ile Leu Phe Leu Lys Leu Ala Leu Phe SerAla His Cys 130 135 140

What is claimed:
 1. An isolated polynucleotide comprising a sequenceselected from the group consisting of: (a) sequences provided in SEQ IDNO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52,53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100,103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140,143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208, and 210-214; (b) complements of the sequencesprovided in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33,35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86,89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134,136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171,174-183, 185, 193-199, 203-206, 208 and 210-214; (c) sequencesconsisting of at least 20 contiguous residues of a sequence provided inSEQ ID NO:1, 2, 5, 9, 10,13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50,52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100,103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140,143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208 and 210-214; (d) sequences that hybridize to asequence provided in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28,32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84,86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128,132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168,171, 174-183, 185, 193-199, 203-206, 208 and 210-214 under moderatelystringent conditions; (e) sequences having at least 75% identity to asequence provided in SEQ ID NO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28,32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84,86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128,132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168,171, 174-183, 185, 193-199, 203-206, 208 and 210-214; (f) sequenceshaving at least 90% identity to a sequence provided in SEQ ID NO:1, 2,5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57,63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111,114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151,156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and210-214 and (g) degenerate variants of a sequence provided in SEQ IDNO:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52,53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100,103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140,143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185,193-199, 203-206, 208 and 210-214.
 2. An isolated polypeptide comprisingan amino acid sequence of an ovarian tumor protein selected from thegroup consisting of: (a) polynucleotides recited in any one of sequencesencoded by a polynucleotide of claim 1; and (b) sequences having atleast 70% identity to a sequence encoded by a polynucleotide of claim 1;and (c) sequences having at least 90% identity to a sequence encoded bya polynucleotide of claim
 1. 3. An expression vector comprising apolynucleotide of claim 1 operably linked to an expression controlsequence.
 4. A host cell transformed or transfected with an expressionvector according to claim
 3. 5. An isolated antibody, or antigen-bindingfragment thereof, that specifically binds to a polypeptide of claim 2.6. A method for detecting the presence of an ovarian cancer in apatient, comprising the steps of: (a) obtaining a biological sample fromthe patient; (b) contacting the biological sample with a binding agentthat binds to a polypeptide of claim 2; (c) detecting in the sample anamount of polypeptide that binds to the binding agent; and (d) comparingthe amount of polypeptide to a predetermined cut-off value and therefromdetermining the presence of a cancer in the patient.
 7. A fusion proteincomprising at least one polypeptide according to claim
 2. 8. Anoligonucleotide that hybridizes to a sequence recited in SEQ ID NO:1, 2,5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57,63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111,114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151,156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and210-214 under moderately stringent conditions.
 9. A method forstimulating and/or expanding T cells specific for a tumor protein,comprising contacting T cells with at least one component selected fromthe group consisting of: (a) polypeptides according to claim 2; (b)polynucleotides according to claim 1; and (c) antigen-presenting cellsthat express a polypeptide according to claim 1, under conditions andfor a time sufficient to permit the stimulation and/or expansion of Tcells.
 10. An isolated T cell population, comprising T cells preparedaccording to the method of claim
 9. 11. A composition comprising a firstcomponent selected from the group consisting of physiologicallyacceptable carriers and immunostimulants, and a second componentselected from the group consisting of: (a) polypeptides according toclaim 2; (b) polynucleotides according to claim 1; (c) antibodiesaccording to claim 5; (d) fusion proteins according to claim 7; (e) Tcell populations according to claim 10; and (f) antigen presenting cellsthat express a polypeptide according to claim
 2. 12. A method forstimulating an immune response in a patient, comprising administering tothe patient a composition of claim
 11. 13. A method for the treatment ofa cancer in a patient, comprising administering to the patient acomposition of claim
 11. 14. A method for determining the presence of acancer in a patient, comprising the steps of: (a) obtaining a biologicalsample from the patient; (b) contacting the biological sample with anoligonucleotide according to claim 8; (c) detecting in the sample anamount of a polynucleotide that hybridizes to the oligonucleotide; and(d) compare the amount of polynucleotide that hybridizes to theoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence of the cancer in the patient.
 15. A diagnostickit comprising at least one oligonucleotide according to claim
 8. 16. Adiagnostic kit comprising at least one antibody according to claim 5 anda detection reagent, wherein the detection reagent comprises a reportergroup.
 17. A method for inhibiting the development of a cancer in apatient, comprising the steps of: (a) incubating CD4+ and/or CD8+ Tcells isolated from a patient with at least one component selected fromthe group consisting of: (i) polypeptides according to claim 2; (ii)polynucleotides according to claim 1; and (iii) antigen presenting cellsthat express a polypeptide of claim 2, such that T cell proliferate; (b)administering to the patient an effective amount of the proliferated Tcells, and thereby inhibiting the development of a cancer in thepatient.
 18. A polypeptide comprising an amino acid sequence selectedfrom the group consisting of sequences recited in SEQ ID NO:200-202,207, 209 and 215.